Composition Comprising Antibodies to LINGO or Fragments Thereof

ABSTRACT

Endogenous LINGO-1 is a negative regulator for neuronal survival, axon regeneration, oligodendrocyte differentiation and myelination. Molecules that block endogenous LINGO-1 function, such anti-LINGO-1 antibodies can be used as therapeutics for the treatment of neuron and oligodendrocyte dysfunction. The present invention provides antibodies specific for LINGO-1, and methods of using such antibodies as antagonists of endogenous LINGO-1 function. The invention further provides specific hybridoma and phage library-derived monoclonal antibodies, nucleic acids encoding these antibodies, and vectors and host cells comprising these antibodies. The invention further provides methods of promoting oligodendrocyte survival and myelination in a vertebrate, comprising administering to a vertebrate in need of such treatment an effective amount of an anti-LINGO-1 antibody

REFERENCE TO RELATED APPLICATIONS

Related applications U.S. Ser. No. 13/243,795, filed Sep. 23, 2011, U.S.Ser. No. 12/500,472, filed Jul. 9, 2009, and U.S. 61/079,355, filed Jul.9, 2008 are herein incorporated by reference in their entireties.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:sequence listing.ascii.txt, Size: 258 kilobytes; and Date of Creation:Jul. 9, 2009) fled with the application is incorporated by reference inits entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to neurology, neurobiology and molecular biology.More particularly, this invention relates to molecules and methods fortreatment of neurological diseases, disorders and injuries such asspinal cord injury.

2. Background of the Invention

Axons and dendrites extend from neurons. The distal tip of an extendingaxon or neurite includes a specialized region, known as the growth cone.Growth cones sense the local environment and guide axonal growth towarda neuron's target cell. Growth cones respond to environmental cues, forexample, surface adhesiveness, growth factors, neurotransmitters andelectric fields. The growth cones generally advance at a rate of one totwo millimeters per day. The growth cone explores the area ahead of itand on either side, by means of elongations classified as lamellipodiaand filopodia. When an elongation contacts an unfavorable surface, itwithdraws. When an elongation contacts a favorable growth surface, itcontinues to extend and guides the growth cone in that direction. Whenthe growth cone reaches an appropriate target cell a synaptic connectionis created.

Nerve cell function is influenced by contact between neurons and othercells in their immediate environment (Rutishauser, et al., 1988,Physiol. Rev. 68:819). These cells include specialized glial cells,oligodendrocytes in the central nervous system (CNS), and Schwann cellsin the peripheral nervous system (PNS), which sheathe the neuronal axonwith myelin (Lemke, 1992, in An Introduction to Molecular Neurobiology,Z. Hall, Ed., p. 281, Sinauer).

CNS neurons have the inherent potential to regenerate after injury, butthey are inhibited from doing so by inhibitory proteins present inmyelin (Brittis et al., 2001, Neuron 30:11-14; Jones et al., 2002, J.Neurosci. 22:2792-2803; Grimpe et al., 2002, J. Neurosci.:22:3144-3160).

Several myelin inhibitory proteins found on oligodendrocytes have beencharacterized. Known examples of myelin inhibitory proteins includeNogoA (Chen et al., Nature, 2000, 403, 434-439; Grandpre et al., Nature2000, 403, 439-444), myelin associated glycoprotein (MAG) (McKerracheret al., 1994, Neuron 13:805-811; Mukhopadhyay et al., 1994, Neuron13:757-767) and oligodendrocyte glycoprotein (OM-gp), Mikol et al.,1988, J. Cell. Biol. 106:1273-1279). Each of these proteins has beenseparately shown to be a ligand for the neuronal Nogo receptor-1 (NgR1(Wang et al., Nature 2002, 417, 941-944; Grandpre et al., Nature 2000,403, 439-444; Chen et al., Nature, 2000, 403, 434-439; Domeniconi etal., Neuron 2002, published online Jun. 28, 2002).

Nogo receptor-1 (NgR1) is a GPI-anchored membrane protein that contains8 leucine rich repeats (Fournier at al., 2001, Nature 409:341-346). Uponinteraction with inhibitory proteins (e.g., NogoA, MAG and OM-gp), theNgR1 complex transduces signals that lead to growth cone collapse andinhibition of neurite outgrowth.

There is an unmet need for molecules and methods for inhibitingNgR1-mediated growth cone collapse and the resulting inhibition ofneurite outgrowth. Additionally there is a need for molecules whichincrease neuronal survival and axon regeneration. Particularly for thetreatment of disease, disorders or injuries which involve axonal injury,neuronal or oligodendrocyte cell death, demyelination or dymyelinationor generally relate to the nervous system.

Such diseases, disorders or injuries include, but are not limited to,multiple sclerosis (MS), progressive multifocal leukoencephalopathy(PML), encephalomyelitis (EPL), central pontine myelolysis (CPM),adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease(PMZ), Globoid cell Leucodystrophy (Krabbe's disease) and WallerianDegeneration, optic neuritis, transverse myelitis, amylotrophic lateralsclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson'sdisease, spinal cord injury, traumatic brain injury, post radiationinjury, neurologic complications of chemotherapy, stroke, acute ischemicoptic neuropathy, vitamin E deficiency, isolated vitamin E deficiencysyndrome, AR, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome,metachromatic leukodystrophy, trigeminal neuralgia, and Bell's palsy.Among these diseases, MS is the most widespread, affecting approximately2.5 million people worldwide.

MS generally begins with a relapsing-remitting pattern of neurologicinvolvement, which then progresses to a chronic phase with increasingneurological damage. MS is associated with the destruction of myelin,oligodendrocytes and axons localized to chronic lesions. Thedemyelination observed in MS is not always permanent and remyelinationhas been documented in early stages of the disease. Remyelination ofneurons requires oligodendrocytes.

Various disease-modifying treatments are available for MS, including theuse of corticosteroids and immunomodulators such as interferon beta andTysabri®. In addition, because of the central role of oligodendrocytesand myelination in MS, there have been efforts to develop therapies toincrease oligodendrocyte numbers or enhance myelination. See, e.g.,Cohen at al., U.S. Pat. No. 5,574,009; Chang et al., N. Engl. J. Med.346: 165-73 (2002). However, there remains an urgent need to deviseadditional therapies for MS and other demyelination and dismyelinationdisorders.

BRIEF SUMMARY OF THE INVENTION

The present invention is based on the discovery that certain LINGO-1antibodies promote survival, proliferation and differentiation ofoligodendrocytes and neuronal cells, as well as myelination of neurons.LINGO-1, previously called Sp3S, has been described in detail inInternational Applications PCT/US2006/026271, filed Jul. 7, 2006,PCT/US2004/008323 filed Mar. 17, 2004, PCT/US2005/022881, filed Jun. 24,2005 and PCT/US2008/000316, filed Jan. 9, 2008, each of which isincorporated by reference in its entirety herein. Based on thesediscoveries, the invention relates generally to antibodies, antigenbinding fragments or derivatives thereof which can be used as anantagonist of LINGO-1. Additionally, the invention generally relates tomethods for treating various diseases, disorders or injuries associatedwith demyelination, dysmyelination, oligodendrocyte/neuronal cell deathor axonal injury by the administration of a LINGO-1 antagonist antibodyor antigen binding fragment.

In certain embodiments, the invention includes an isolated antibody orantigen binding fragment thereof which specifically binds to the sameLINGO-1 epitope as the reference monoclonal antibody Li62 or Li81.

Certain embodiments of the invention include an isolated polypeptidecomprising an immunoglobulin heavy chain variable region (VH) whereinthe CDR1, CDR2 and CDR3 regions are selected from the polypeptidesequences shown in Table 3 or at least 80%, 85%, 90% or 95% identical tothe polypeptide sequences shown in Table 3 or at least 80%, 85%, 90, 95%or 100% identical to the VH CDR1, CDR2 and CDR3 regions of theimmunoglobulin heavy chain of Li62 or Li81. In some embodiments, the VHcomprises the polypeptide sequence of SEQ ID NO: 4 or SEQ ID NO:8 or anyone of SEQ ID NOs: 17 to 49.

Certain embodiments of the invention include an isolated polypeptidecomprising an immunoglobulin light chain variable region (VL) whereinthe CDR1, CDR2 and CDR3 regions are selected from the polypeptidesequences shown in Table 4 or at least 80%, 85%, 90% or 95% identical tothe polypeptide sequences shown in Table 4 or at least 80%, 85%, 90%,95% or 100% identical to the VL CDR1, CDR2 and CDR3 regions of theimmunoglobulin light chain of Li62 or Li81.

Certain embodiments of the invention include an isolated polypeptidecomprising an immunoglobulin heavy chain variable region (VH) selectedfrom the group consisting of SEQ ID NOs: 1, 5, and 53-85 or at least80%, 85%, 90% or 95% identical to said SEQ ID NOs: 1, 5 and 53-85.

Certain embodiments of the invention include an isolated polypeptidecomprising an immunoglobulin light chain variable region (VL) selectedfrom the group consisting of SEQ ID NOs: 9 and 13, as shown in Table 4,or at least 80%, 85%, 90% or 95% identical to said SEQ ID NOs: 9 and 13,as shown in Table 4.

Other embodiments of the invention include an isolated polynucleotidecomprising a nucleic acid encoding an immunoglobulin heavy chainvariable region (VH) selected from the group consisting of SEQ ID NOs:1, 5 and 53-85, or at least 80%, 85%, 90% or 95% identical to said SEQID NOs: 1, 5 and 53-85. In some embodiments, the polynucleotidecomprises a nucleic acid encoding the polypeptide sequence of SEQ ID NO:4 or SEQ ID NO:8 or any one of SEQ ID NOs: 17 to 49.

Other embodiments of the invention include an isolated polynucleotidecomprising a nucleic acid encoding an immunoglobulin light chainvariable region (VL) selected from the group consisting of SEQ ID NOs: 9and 13, as shown in Table 4, or at least 80%, 85%, 90% or 95% identicalto said SEQ ID NOs: 9 and 13, as shown in Table 4.

In certain embodiments, the invention includes compositions comprisingthe antibodies or antigen binding fragments described herein.

In additional embodiments, the invention includes methods for treatingCNS injury, ALS, Huntington's disease, Alzheimer's disease, Parkinson'sdisease, diabetic neuropathy and stroke comprising administering to ananimal in need of said treatment an effective amount of an agentselected from the group consisting of an isolated LINGO-1 antibody orfragment thereof or compositions comprising said antibody or fragmentthereof.

In other embodiments, the invention includes methods for treatingdiseases or disorders associated with inhibition of oligodendrocytegrowth or differentiation; demyelination or dysmyelination of CNSneurons including multiple sclerosis (MS), progressive multifocalleukoencephalopathy (PML), encephalomyelitis (EPL), central pontinemyelolysis (CPM), Wallerian Degeneration, adrenoleukodystrophy,Alexander's disease, and Pelizaeus Merzbacher disease (PMZ) byadministering to an animal in need of said treatment an effective amountof an agent selected from the group consisting of an isolated LINGO-1antibody or fragment thereof or compositions comprising said antibody orfragment thereof.

Other embodiments of the present invention include a method ofinhibiting signal transduction by Nogo receptor 1 (NgR1), comprisingcontacting the NgR1 with an effective amount of an agent selected fromthe group consisting of the isolated LINGO-1 antibody or fragmentthereof or compositions comprising said antibody or fragment thereof.

Additional embodiments of the present invention include a method ofdecreasing inhibition of axonal growth of a central nervous system (CNS)neuron, comprising contacting the neuron with an effective amount of anagent selected from the group consisting of the isolated LINGO-1antibody or fragment thereof of or compositions comprising said antibodyor fragment thereof.

Other embodiments of the present invention include a method ofinhibiting growth cone collapse of a CNS neuron, comprising contactingthe neuron with an effective amount of an agent selected from the groupconsisting of the isolated LINGO-1 antibody or fragment thereof orcompositions comprising said antibody or fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1: Western blot of co-cultured oligodendrocyte precursor cells andDRGs after incubation with anti-LINGO-1 antibodies (Li33 PDL, Li62(agly) and Li81 (agly)) and control antibody (h5C8) as described inExample 2.

FIG. 2: Western blot of MBP, MOG and β-actin in rat A2B5+ progenitorcells treated with anti-LINGO-1 antibody (Li81 (agly)) or controlantibody (Ctrl) as described in Example 4.

FIG. 3: Bar graph showing the number of MBP-positive cells in humanoligodendrocyte precursor cell cultures treated with anti-LINGO-1antibody (Li81 (agly)) or a control antibody (hIgG1) as described inExample 5.

FIG. 4: Bar graph showing intensity of black gold immunostaining to markmyelination in lysolecithin (“Lyso”)-treated brain slices exposed tocontrol antibody (5C8) or anti-LINGO-1 antibody (Li81 (agly)) atconcentrations of 30 μg/ml (81-30), 10 μg/ml (81-10), 3 μg/ml (81-3), or1 μg/ml (81-1). Experiments were performed as described in Example 6.

FIG. 5: Graphs depicting IC50 values of an aglycosylated anti-LINGO-1antibody (Li81 (agly)), a control antibody (WT 5c8) and an aglycosylatedcontrol antibody (agly 5c8) for human Fc receptors CD16 (A), CD32a (B),CD32b (C) and CD64 (D). Experiments were performed as described inExample 7.

FIG. 6: Graph depicting binding of anti-LINGO-1 antibodies (Li33(“Di33IgG1”) and Li13 “Di13IgG1”)), aglycosylated anti-LINGO-1 antibody(Li81 (agly) (“Di81IgG1Agly”), and a control antibody (huIgG1) to CD64and CD32 as measured by the cell bridging assay described in Example 7.

FIG. 7: Graph depicting complement activation in CHO cells expressingLINGO-1 incubated with anti-LINGO-1 antibodies (Li81 (agly) (“Dli81”),Li33 (“Dli33IgG1”), Li13 (“Dli13IgG1”)) and a positive control antibody(LTbetaR-Ig). Experiments were performed as described in Example 8.

FIG. 8: Images showing lesions in lysolecithin-treated animalsadministered a control antibody (“Ctrl”) or an anti-LINGO-1 antibody(Li81 (agly)) and a bar graph depicting the size of demyelinated lesionsin rats treated with lysolecithin and administered control antibody oranti-LINGO-1 antibody (Li81 (agly)). Experiments were, performed asdescribed in Example 9.

FIG. 9: Graph depicting EAE score to assess paralysis in ratsadministered recombinant myelin oligodendrocyte glycoprotein and treatedwith control antibody (“Isotype ctr”) or anti-LINGO-1 antibody (Li81).Downward arrows indicate timepoints at which antibody treatment wasadministered. Experiments were performed as described in Example 10.

FIG. 10: Graph showing the binding of Li113 Fab to LINGO-1 as measuredby ELISA assay.

FIG. 11: Graph depicting efficacy of LINGO-1 monoclonal antibodies andFabs in an oligodendrocyte differentiation assay. Bar height representsthe concentration of MBP as measured by ELISA. Antibodies were tested atconcentrations of 1 μg/ml (black), 0.3 μg/ml (dark grey), 0.1 μg/ml(light grey) and 0.03 μg/ml (white).

FIG. 12: Image showing the size exclusion chromatography profiles forLi33 Ig1 (agly) and Ig2. Top panel shows the elution profile of BIO RADgel filtration markers and shows molecular masses.

FIG. 13: Graph depicting the denaturation of Li33 Ig1 and Ig2 byguanidine hydrochloride. Flourescence data from the emission spectra at350 nm are plotted as a function of the guanidine hydrochlorideconcentration and standardized using the change in flourecence frommaximum for each test condition. NEM refers to N-ethylmaleimide and TCEPrefers to Tris(2-carboxyethyl)phosphine.

FIG. 14: Image showing the results of an analytical ultracentrifugationevaluating the aggregation state of Li33 Ig2. Absorbance scan data fromvelocity sedimentation centrifugation studies with Li33 Ig2 Mab at 0.4mg/ml (A), 7 mg/ml (B) and 27 mg/ml (C) are shown. Top panels show rawabsorbance data as a function of time. Bottom panels show relativeconcentrations as a function of sedimentation coefficient.

FIG. 15: Image showing protein-protein interactions in the Li33 Fabstructure.

FIG. 16: Image depicting methods of generating PEGylated Fabs throughdirect expression.

FIG. 17: Image displaying an SDS-PAGE gel under non-reducing conditionsshowing the results of PEGylated Fab direct expression studies using themethods shown in FIG. 16. Arrowhead indicates PEGylated Fab.

FIG. 18: Image displaying an SDS-PAGE gel under non-reducing conditionsshowing the results of PEGylated Fab enzymatic digestion studies. Lane 1shows molecular mass markers. Lane 2 shows Li33 Ig1 Mab. Lane 3 showsLi33 Ig1 Fab2. Lane 4 shows Li33 Ig1 Fab2 treated with TCEP, and lane 5shows Li33 Ig1 Fab2 treated with TCEP and then with PEG. Arrow indicatesPEGylated Li33 Fab′ product.

FIG. 19: Graph depicting the results of a FACS assay to assess bindingof PEGylated LINGO-1 antibodies to LINGO-1.

FIG. 20: Graph depicting the results of Li81 binding measured in adirect binding ELISA assay using LINGO-1 coated plates.

FIG. 21: Graph depicting results of an oligodendrocyte differentiationassay using Li81. Bar height indicates the concentration of MBP asmeasured by ELISA. Li81 RTP-RO8 indicates an Li81 (agly) referencestandard.

FIG. 22: Graph depicting results of remyelination assay using Li81antibody and antibody fragments. Bar height represents the intensity ofblack gold signal.

FIG. 23: Graphs depicting results of thermal denaturation studies ofLINGO-1 antibodies and antibody fragments. Bar height indicates TM.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a LINGO-1 antibody,” is understood torepresent one or more LINGO-1 antibodies. As such, the terms “a” (or“an”), “one or more,” and “at least one” can be used interchangeablyherein.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

Also included as polypeptides of the present invention are fragments,derivatives, analogs, or variants of the foregoing polypeptides, and anycombination thereof. The terms “fragment,” “variant,” “derivative” and“analog” when referring to LINGO-1 antibodies or antibody polypeptidesof the present invention include any polypeptides which retain at leastsome of the antigen-binding properties of the corresponding nativeantibody or polypeptide. Fragments of polypeptides of the presentinvention include proteolytic fragments, as well as deletion fragments,in addition to specific antibody fragments discussed elsewhere herein.Variants of LINGO-1 antibodies and antibody polypeptides of the presentinvention include fragments as described above, and also polypeptideswith altered amino acid sequences due to amino acid substitutions,deletions, or insertions. Variants may occur naturally or benon-naturally occurring Non-naturally occurring variants may be producedusing art-known mutagenesis techniques. Variant polypeptides maycomprise conservative or non-conservative amino acid substitutions,deletions or additions. Derivatives of LINGO-1 antibodies and antibodypolypeptides of the present invention, are polypeptides which have beenaltered so as to exhibit additional features not found on the nativepolypeptide. Examples include fusion proteins. Variant polypeptides mayalso be referred to herein as “polypeptide analogs.” As used herein a“derivative” of a LINGO-1 antibody or antibody polypeptide refers to asubject polypeptide having one or more residues chemically derivatizedby reaction of a functional side group. Also included as “derivatives”are those peptides which contain one or more naturally occurring aminoacid derivatives of the twenty standard amino acids. For example,4-hydroxyproline may be substituted for proline; 5-hydroxylysine may besubstituted for lysine; 3-methylhistidine may be substituted forhistidine; homoserine may be substituted for serine; and ornithine maybe substituted for lysine.

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide may comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refer to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encoding aLINGO-1 antibody contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding aLINGO-1 antibody or fragment, variant, or derivative thereof.Heterologous coding regions include without limitation specializedelements or motifs, such as a secretory signal peptide or a heterologousfunctional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid whichencodes a polypeptide normally may include a promoter and/or othertranscription or translation control elements operably associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between the twoDNA fragments does not interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product orinterfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter may be a cell-specificpromoter that directs substantial transcription of the DNA only inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence which is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucuronidase.

The present invention is directed to certain LINGO-1 antibodies, orantigen-binding fragments, variants, or derivatives thereof. Unlessspecifically referring to full-sized antibodies such asnaturally-occurring antibodies, the term “LINGO-1 antibodies”encompasses full-sized antibodies as well as antigen-binding fragments,variants, analogs, or derivatives of such antibodies, e.g., naturallyoccurring antibody or immunoglobulin molecules or engineered antibodymolecules or fragments that bind antigen in a manner similar to antibodymolecules.

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin comprises at least the variabledomain of a heavy chain, and normally comprises at least the variabledomains of a heavy chain and a light chain. Basic immunoglobulinstructures in vertebrate systems are relatively well understood. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988).

As will be discussed in more detail below, the term “immunoglobulin”comprises various broad classes of polypeptides that can bedistinguished biochemically. Those skilled in the art will appreciatethat heavy chains are classified as gamma, mu, alpha, delta, or epsilon,(γ, μ, α, δ, ε) with some subclasses among them (e.g., γ1-γ4). It is thenature of this chain that determines the “class” of the antibody as IgG,IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses(isotypes) e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, etc. are wellcharacterized and are known to confer functional specialization.Modified versions of each of these classes and isotypes are readilydiscernable to the skilled artisan in view of the instant disclosureand, accordingly, are within the scope of the instant invention. Allimmunoglobulin classes are clearly within the scope of the presentinvention, the following discussion will generally be directed to theIgG class of immunoglobulin molecules. With regard to IgG, a standardimmunoglobulin molecule comprises two identical light chain polypeptidesof molecular weight approximately 23,000 Daltons, and two identicalheavy chain polypeptides of molecular weight 53,000-70,000. The fourchains are typically joined by disulfide bonds in a “Y” configurationwherein the light chains bracket the heavy chains starting at the mouthof the “Y” and continuing through the variable region.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (V_(L)) and heavy (V_(H)) chain portionsdetermine antigen recognition and specificity. Conversely, the constantdomains of the light chain (C_(L)) and the heavy chain (C_(H)1, C_(H)2or C_(H)3) confer important biological properties such as secretion,transplacental mobility, Fc receptor binding, complement binding, andthe like. By convention the numbering of the constant region domainsincreases as they become more distal from the antigen binding site oramino-terminus of the antibody. The N-terminal portion is a variableregion and at the C-terminal portion is a constant region; the C_(H)3and C_(L) domains actually comprise the carboxy-terminus of the heavyand light chain, respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the V_(L) domain and V_(H) domain, or subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three dimensional antigen binding site. Thisquaternary antibody structure forms the antigen binding site present atthe end of each arm of the Y. More specifically, the antigen bindingsite is defined by three CDRs on each of the V_(H) and V_(L) chains. Insome instances, e.g., certain immunoglobulin molecules derived fromcamelid species or engineered based on camelid immunoglobulins, acomplete immunoglobulin molecule may consist of heavy chains only, withno light chains. See, e.g., Hammers-Casterman at al., Nature 363:446-448(1993):

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen binding domain are short,non-contiguous sequences of amino acids that are specifically,positioned to form the antigen binding domain as the antibody assumesits three dimensional configuration in an aqueous environment. Theremainder of the amino acids in the antigen binding domains, referred toas “framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops which connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable regionby one of ordinary skill in the art, since they have been preciselydefined (see, “Sequences of Proteins of Immunological Interest,” Kabat,E., et al., U.S. Department of Health and Human Services, (1983); andChothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which areincorporated herein by reference in their entireties).

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaet al., J. Mol. Biol. 196:901-917 (1987), which are incorporated hereinby reference, where the definitions include overlapping or subsets ofamino acid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein. The appropriate amino acid residues whichencompass the CDRs as defined by each of the above cited references areset forth below in Table I as a comparison. The exact residue numberswhich encompass a particular CDR will vary depending on the sequence andsize of the CDR. Those skilled in the art can routinely determine whichresidues comprise a particular CDR given the variable region amino acidsequence of the antibody.

TABLE 1 CDR Definitions¹ Kabat Chothia V_(H) CDR1 31-35 26-32 V_(H) CDR250-65 52-58 V_(H) CDR3  95-102  95-102 V_(L) CDR1 24-34 26-32 V_(L) CDR250-56 50-52 V_(L) CDR3 89-97 91-96 ¹Numbering of all CDR definitions inTable 1 is according to the numbering conventions set forth by Kabat etal. (see below).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambigously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in a LINGO-1 antibody or antigen-bindingfragment, variant, or derivative thereof of the present invention areaccording to the Kabat numbering system.

In camelid species, the heavy chain variable region, referred to asV_(H)H, forms the entire antigen-binding domain. The main differencesbetween camelid V_(H)H variable regions and those derived fromconventional antibodies (V_(H)) include (a) more hydrophobic amino acidsin the light chain contact surface of V_(H) as compared to thecorresponding region in V_(H)H, (b) a longer CDR3 in V_(H)H, and (c) thefrequent occurrence of a disulfide bond between CDR1 and CDR3 in V_(H)H.

Antibodies or antigen-binding fragments, variants, or derivativesthereof of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized, primatized, or chimericantibodies, single chain antibodies, epitope-binding fragments, e.g.,Fab, Fab′ and F(ab′)₂, Fd, Fvs, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv), fragments comprising either aV_(L) or V_(H) domain, fragments produced by a Fab expression library,and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Idantibodies to LINGO-1 antibodies disclosed herein). ScFv molecules areknown in the art and are described, e.g., in U.S. Pat. No. 5,892,019.Immunoglobulin or antibody molecules of the invention can be of any type(e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Antibody fragments, including single-chain antibodies, may comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, C_(H)1, C_(H)2, and C_(H)3domains. Also included in the invention are antigen-binding fragmentsalso comprising any combination of variable region(s) with a hingeregion, C_(H)1, C_(H)2, and C_(H)3 domains. Antibodies or immunospecificfragments thereof for use in the diagnostic and therapeutic methodsdisclosed herein may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine, donkey, rabbit,goat, guinea pig, camel, llama, horse, or chicken antibodies. In anotherembodiment, the variable region may be condricthoid in origin (e.g.,from sharks). As used herein, “human” antibodies include antibodieshaving the amino acid sequence of a human immunoglobulin and includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example in, U.S.Pat. No. 5,939,598 by Kucherlapati et al.

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a C_(H)1domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a C_(H)2 domain, a C_(H)3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention may comprise apolypeptide chain comprising a C_(H)1 domain; a polypeptide chaincomprising a C_(H)1 domain, at least a portion of a hinge domain, and aC_(H)2 domain; a polypeptide chain comprising a C_(H)1 domain and aC_(H)3 domain; a polypeptide chain comprising a C_(H)1 domain, at leasta portion of a hinge domain, and a C_(H)3 domain, or a polypeptide chaincomprising a C_(H)1 domain, at least a portion of a hinge domain, aC_(H)2 domain, and a C_(H)3 domain. In another embodiment, a polypeptideof the invention comprises a polypeptide chain comprising a C_(H)3domain. Further, a binding polypeptide for use in the invention may lackat least a portion of a C_(H)2 domain (e.g., all or part of a C_(H)2domain). As set forth above, it will be understood by one of ordinaryskill in the art that these domains (e.g., the heavy chain portions) maybe modified such that they vary in amino acid sequence from thenaturally occurring immunoglobulin molecule.

In certain LINGO-1 antibodies, or antigen-binding fragments, variants,or derivatives thereof disclosed herein, the heavy chain portions of onepolypeptide chain of a multimer are identical to those on a secondpolypeptide chain of the multimer. Alternatively, heavy chainportion-containing monomers of the invention are not identical. Forexample, each monomer may comprise a different target binding site,forming, for example, a bispecific antibody.

The heavy chain portions of a binding polypeptide for use in thediagnostic and treatment methods disclosed herein may be derived fromdifferent immunoglobulin molecules. For example, a heavy chain portionof a polypeptide may comprise a C_(H)1 domain derived from an IgG1molecule and a hinge region derived from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a hinge region derived, inpart, from an IgG 1 molecule and, in part, from an IgG3 molecule. Inanother example, a heavy chain portion can comprise a chimeric hingederived, in part, from an IgG1 molecule and, in part, from an IgG4molecule.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. Preferably, thelight chain portion comprises at least one of a V_(L) or C_(L) domain.

LINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein may be described or specified interms of the epitope(s) or portion(s) of an antigen, e.g., a targetpolypeptide (LINGO-1) that they recognize or specifically bind. Theportion of a target polypeptide which specifically interacts with theantigen binding domain of an antibody is an “epitope,” or an “antigenicdeterminant.” A target polypeptide may comprise a single epitope, buttypically comprises at least two epitopes, and can include any number ofepitopes, depending on the size, conformation, and type of antigen.Furthermore, it should be noted that an “epitope” on a targetpolypeptide may be or include non-polypeptide elements, e.g., an“epitope may include a carbohydrate side chain.

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about tour to five amino acids. Peptide or polypeptideepitopes preferably contain at least seven, more preferably at leastnine and most preferably between at least about 15 to about 30 aminoacids. Since a CDR can recognize an antigenic peptide or polypeptide inits tertiary form, the amino acids comprising an epitope need not becontiguous, and in some cases, may not even be on the same peptidechain. In the present invention, peptide or polypeptide epitoperecognized by LINGO-1 antibodies of the present invention contains asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 15, at least20, at least 25, or between about 15 to about 30 contiguous ornon-contiguous amino acids of LINGO-1.

By “specifically binds,” it is generally meant that an antibody binds toan epitope via its antigen binding domain, and that the binding entailssome complementarity between the antigen binding domain and the epitope.According to this definition, an antibody is said to “specifically bind”to an epitope when it binds to that epitope, via its antigen bindingdomain more readily than it would bind to a random, unrelated epitope.The term “specificity” is used herein to qualify the relative affinityby which a certain antibody binds to a certain epitope. For example,antibody “A” may be deemed to have a higher specificity for a givenepitope than antibody “B,” or antibody “A” may be said to bind toepitope “C” with a higher specificity than it has for related epitope“D.”

By “preferentially binds,” it is meant that the antibody specificallybinds to an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope. Thus, an antibody which“preferentially binds” to a given epitope would more likely bind to thatepitope than to a related epitope, even though such an antibody maycross-react with the related epitope.

By way of non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds said first epitope with adissociation constant (K_(D)) that is less than the antibody's K_(D) forthe second epitope. In another non-limiting example, an antibody may beconsidered to bind a first antigen preferentially if it binds the firstepitope with an affinity that is at least one order of magnitude lessthan the antibody's K_(D) for the second epitope. In anothernon-limiting example, an antibody may be considered to bind a firstepitope preferentially if it binds the first epitope with an affinitythat is at least two orders of magnitude less than the antibody's K_(D)for the second epitope.

in another non-limiting, example, an antibody may be considered to binda first epitope preferentially if it binds the first epitope with an offrate (k(off) that is less than the antibody's k(off) for the secondepitope. In another non-limiting example, an antibody may be consideredto bind a first epitope preferentially if it binds the first epitopewith an affinity that is at least one order of magnitude less than theantibody's k(off) for the second epitope. In another non-limitingexample, an antibody may be considered to bind a first epitopepreferentially if it binds the first epitope with an affinity that is atleast two orders of magnitude less than the antibody's k(off) for thesecond epitope.

An antibody or or antigen-binding fragment, variant, or derivativedisclosed herein may be said to bind a target polypeptide disclosedherein or a fragment or variant thereof with an off rate (k(off)) ofless than or equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹ or 10⁻³sec⁻¹. More preferably, an antibody of the invention may be said to binda target polypeptide disclosed herein or a fragment or variant thereofwith an off rate (k(off)) less than or equal to 5×10⁻⁴ sec⁻¹, 10⁻⁴sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹ 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷sec⁻¹ or 10⁻⁷ sec⁻¹.

An antibody or or antigen-binding fragment, variant, or derivativedisclosed herein may be said to bind a target polypeptide disclosedherein or a fragment or variant thereof with an on rate (k(on)) ofgreater than or equal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹or 5×10⁴ M⁻¹ sec⁻¹. More preferably, an antibody of the invention may besaid to bind a target polypeptide disclosed herein or a fragment orvariant thereof with an on rate (k(on)) greater than or equal to 10⁵ M⁻¹sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×10⁶ M⁻¹ sec⁻¹ or 10⁷ M⁻¹sec⁻¹.

An antibody is said to competitively inhibit binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody to the epitope. Competitive inhibition may be determined by anymethod known in the art, for example, competition ELISA assays. Anantibody may be said to competitively inhibit binding of the referenceantibody to a given epitope by at least 90%, at least 80%, at least 70%,at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of animmunoglobulin molecule. See, e.g., Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988)at pages 27-28. As used herein, the term “avidity” refers to the overallstability of the complex between a population of immunoglobulins and anantigen, that is, the functional combining strength of an immunoglobulinmixture with the antigen. See, e.g., Harlow at pages 29-34. Avidity isrelated to both the affinity of individual immunoglobulin molecules inthe population with specific epitopes, and also the valencies of theimmunoglobulins and the antigen. For example, the interaction between abivalent monoclonal antibody and an antigen with a highly repeatingepitope structure, such as a polymer, would be one of high avidity.

LINGO-1 antibodies or antigen-binding fragments, variants or derivativesthereof of the invention may also be described or specified in terms oftheir cross-reactivity. As used herein, the term “cross-reactivity”refers to the ability of an antibody, specific for one antigen, to reactwith a second antigen; a measure of relatedness between two differentantigenic substances. Thus, an antibody is cross reactive if it binds toan epitope other than the one that induced its formation. The crossreactive epitope generally contains many of the same complementarystructural features as the inducing epitope, and in some cases, mayactually fit better than the original.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

LINGO-1 antibodies or antigen-binding fragments, variants or derivativesthereof of the invention may also be described or specified in terms oftheir binding affinity to a polypeptide of the invention. Preferredbinding affinities include those with a dissociation constant or Kd lessthan 5×10⁻²M, 10⁻² M, 5×10⁻³M, 10⁻³M, 5×10⁻⁴ M, 10⁻⁴M, 5×10⁻⁵M, 10⁻⁵M,5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁴ M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹ M,5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M, 10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M,5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

LINGO-1 antibodies or antigen-binding fragments, variants or derivativesthereof of the invention may be “multispecific,” e.g., bispecific,trispecific or of greater multispecificity, meaning that it recognizesand binds to two or more different epitopes present on one or moredifferent antigens (e.g., proteins) at the same time. Thus, whether aLINGO-1 antibody is “monospecific” or “multispecific,” e.g.,“bispecific,” refers to the number of different epitopes with which abinding polypeptide reacts. Multispecific antibodies may be specific fordifferent epitopes of a target polypeptide described herein or may bespecific for a target polypeptide as well as for a heterologous epitope,such as a heterologous polypeptide or solid support material.

As used herein the term “valency” refers to the number of potentialbinding domains, e.g., antigen binding domains, present in a LINGO-1antibody, binding polypeptide or antibody. Each binding domainspecifically binds one epitope. When a LINGO-1 antibody, bindingpolypeptide or antibody comprises more than one binding domain, eachbinding domain may specifically bind the same epitope, for an antibodywith two binding domains, termed “bivalent monospecific,” or todifferent epitopes, for an antibody with two binding domains, termed“bivalent bispecific.” An antibody may also be bispecific and bivalentfor each specificity (termed “bispecific tetravalent antibodies”). Inanother embodiment, tetravalent minibodies or domain deleted antibodiescan be made.

Bispecific bivalent antibodies, and methods of making them, aredescribed, for instance in U.S. Pat. Nos. 5,731,168; 5,807,706;5,821,333; and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537, thedisclosures of all of which are incorporated by reference herein.Bispecific tetravalent antibodies, and methods of making them aredescribed, for instance, in WO 02/096948 and WO 00/44788, thedisclosures of both of which are incorporated by reference herein. Seegenerally, PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO92/05793; Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos.4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al.,J. Immunol. 148:1547-1553 (1992).

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “V_(H) domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “C_(H)1 domain” includes the first (most amino terminal)constant region domain of an immunoglobulin heavy chain. The C_(H)1domain is adjacent to the V_(H) domain and is amino terminal to thehinge region of an immunoglobulin heavy chain molecule.

As used herein the term “C_(H)2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat E A et al. op. cit. The C_(H)2 domain is unique in that it isnot closely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two C_(H)2 domains of anintact native IgG molecule. It is also well documented that the C_(H)3domain extends from the C_(H)2 domain to the C-terminal of the IgGmolecule and comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the C_(H)1 domain to the C_(H)2 domain. Thishinge region comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al., J.Immunol. 161:4083 (1998)).

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the C_(H)1 and C_(L)regions are linked by a disulfide bond and the two heavy chains arelinked by two disulfide bonds at positions corresponding to 239 and 242using the Kabat numbering system (position 226 or 229, EU numberingsystem).

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which may beintact, partial or modified in accordance with the instant invention) isobtained from a second species. In preferred embodiments the targetbinding region or site will be from a non-human source (e.g. mouse orprimate) and the constant region is human.

As used herein, the term “engineered antibody” refers to an antibody inwhich the variable domain in either the heavy and light chain or both isaltered by at least partial replacement of one or more CDRs from anantibody of known specificity and, if necessary, by partial frameworkregion replacement and sequence changing. Although the CDRs may bederived from an antibody of the same class or even subclass as theantibody from which the framework regions are derived, it is envisagedthat the CDRs will be derived from an antibody of different class andpreferably from an antibody from a different species. An engineeredantibody in which one or more “donor” CDRs from a non-human antibody ofknown specificity is grafted into a human heavy or light chain frameworkregion is referred to herein as a “humanized antibody.” It may not benecessary to replace all of the CDRs with the complete CDRs from thedonor variable region to transfer the antigen binding capacity of onevariable domain to another. Rather, it may only be necessary to transferthose residues that are necessary to maintain the activity of the targetbinding site. Given the explanations set forth in, e.g., U.S. Pat. Nos.5,585,089, 5,693,761, 5,693,762, and 6,180,370, it will be well withinthe competence of those skilled in the art, either by carrying outroutine experimentation or by trial and error testing to obtain afunctional engineered or humanized antibody.

As used herein the term “properly folded polypeptide” includespolypeptides (e.g., LINGO-1 antibodies) in which all of the functionaldomains comprising the polypeptide are distinctly active. As usedherein, the term “improperly folded polypeptide” includes polypeptidesin which at least one of the functional domains of the polypeptide isnot active. In one embodiment, a properly folded polypeptide comprisespolypeptide chains linked by at least one disulfide bond and,conversely, an improperly folded polypeptide comprises polypeptidechains not linked by at least one disulfide bond.

As used herein the term “engineered” includes manipulation of nucleicacid or polypeptide molecules by synthetic means (e.g. by recombinanttechniques, in vitro peptide synthesis, by enzymatic or chemicalcoupling of peptides or some combination of these techniques).

As used herein, the terms “linked,” “fused” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two ore more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature.) Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence. Forexample, polynucleotides encoding the CDRs of an immunoglobulin variableregion may be fused, in-frame, but be separated by a polynucleotideencoding at least one immunoglobulin framework region or additional CDRregions, as long as the “fused” CDRs are co-translated as part of acontinuous polypeptide.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminal direction in which residues that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. The processincludes any manifestation of the functional presence of the gene withinthe cell including, without limitation, gene knockdown as well as bothtransient expression and stable expression. It includes withoutlimitation transcription of the gene into messenger RNA (mRNA), transferRNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) orany other RNA product, and the translation of such mRNA intopolypeptide(s). If the final desired product is a biochemical,expression includes the creation of that biochemical and any precursors.Expression of a gene produces a “gene product.” As used herein, a geneproduct can be either a nucleic acid, e.g., a messenger RNA produced bytranscription of a gene, or a polypeptide which is translated from atranscript. Gene products described herein further include nucleic acidswith post transcriptional modifications, e.g., polyadenylation, orpolypeptides with post translational modifications, e.g., methylation,glycosylation, the addition of lipids, association with other proteinsubunits, proteolytic cleavage, and the like.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the progression of multiplesclerosis. Beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sports, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, andso on.

As used herein, phrases such as “a subject that would benefit fromadministration of a LINGO-1 antibody” and “an animal in need oftreatment” includes subjects, such as mammalian subjects, that wouldbenefit from administration of a LINGO-1 antibody used, e.g., fordetection of a LINGO-1 polypeptide (e.g., for a diagnostic procedure)and/or from treatment, i.e., palliation or prevention of a disease suchas MS, with a LINGO-1 antibody. As described in more detail herein, theLINGO-1 antibody can be used in unconjugated form or can be conjugated,e.g., to a drug, prodrug, or an isotope.

II. LINGO-1

Naturally occurring humanLINGO-1 (LINGO-1) is a glycosylated centralnervous system-specific protein which is predicted to have 614 aminoacids (SEQ ID NO: 51), including a 33 amino acid signal sequence. Asused herein, the term “LINGO-1” is used interchangeably with the term“Sp35” as described in International Applications PCT/US2006/026271,filed Jul. 7, 2006, PCT/US2004/008323, filed Mar. 17, 2004,PCT/US2005/022881, filed Jun. 24, 2005 and PCT/US2008/000316, filed Jan.9, 2008, each of which is incorporated herein by reference in itsentirety. LINGO-1 is also known in the art by the names LRRN6, LRRN6A,FLJ14594, LERN1, MGC17422 and UNQ201. The human, full-length wild-typeLINGO-1 polypeptide contains an LRR domain consisting of 14 leucine-richrepeats (including N- and C-terminal caps), an Ig domain, atransmembrane region, and a cytoplasmic domain. The cytoplasmic domaincontains a canonical tyrosine phosphorylation site. In addition, thenaturally occurring LINGO-1 protein contains a signal sequence, a shortbasic region between the LRRCT and Ig domain, and a transmembrane regionbetween the Ig domain and the cytoplasmic domain. The human LINGO-1 gene(SEQ ID NO:52) contains alternative translation start codons, so thatsix additional amino acids, i.e., MQVSKR (SEQ ID NO:87) may or may notbe present at the N-terminus of the LINGO-1 signal sequence. Table 2lists the LINGO-1 domains and other regions, according to amino acidresidue number, based on the LINGO-1 amino acid sequence, presentedherein as SEQ ID NO: 51. The LINGO-1 polypeptide is characterized inmore detail in PCT Publication No. WO 2004/085648, which is incorporatedherein by reference in its entirety.

TABLE 2 LINGO-1 Domains Domain or Region Beginning Residue EndingResidue Signal Sequence  1 33 or 35 LRRNT 34 or 36  64 LRR  66  89 LRR 90 113 LRR 114 137 LRR 138 161 LRR 162 185 LRR 186 209 LRR 210 233 LRR234 257 LRR 258 281 LRR 282 305 LRR 306 329 LRR 330 353 LRRCT 363 414 or416 Basic 415 or 417 424 Ig 419 493 Connecting sequence 494 551Transmembrane 552 576 Cytoplasmic 577 614

Tissue distribution and developmental expression of LINGO-1 has beenstudied in humans and rats. LINGO-1 biology has been studied in anexperimental animal (rat) model. Expression of rat LINGO-1 is localizedto neurons and oligodendrocytes, as determined by northern blot andimmuno-histochemical staining. Rat LINGO-1 mRNA expression level isregulated developmentally, peaking shortly after birth, i.e., ca.postnatal day one. In a rat spinal cord transection injury model,LINGO-1 is up-regulated at the injury site, as determined by RT-PCR. SeeMi et al. Nature Neurosci. 7:221-228 (2004).

In the context of the amino acids comprising the various structural andfunctional domains of a LINGO-1 polypeptide, the term “about” includesthe particularly recited value and values larger or smaller by several(e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) amino acids. Since the locationof these domains as listed in Table 2 have been predicted by computergraphics, one of ordinary skill would appreciate that the amino acidresidues constituting the domains may vary slightly (e.g., by about 1 to15 residues) depending on the criteria used to define the domain.

The inventors have discovered that full-length, wild-type LINGO-1 bindsto NgR1. See PCT Publication No. WO 2004/085648. The inventors have alsodiscovered that LINGO-1 is expressed in oligodendrocytes and that theLINGO-1 protein is involved in the regulation ofoligodendrocyte-mediated myelination of axons. See U.S PatentPublication No. 2006/0009388 A1, which is incorporated herein byreference in its entirety.

The nucleotide sequence for the full-length LINGO-1 molecule is asfollows:

(SEQ ID NO: 52)ATGCTGGCGGGGGGCGTGAGGAGCATGCCCAGCCCCCTCCTGGCCTGCTGGCAGCCCATCCTCCTGCTGGTGCTGGGCTCAGTGCTGTCAGGCTCGGCCACGGGCTGCCCGCCCCGCTGCGAGTGCTCCGCCCAGGACCGCGCTGTGCTGTGCCACCGCAAGCGCTTTGTGGCAGTCCCCGAGGGCATCCCCACCGAGACGCGCCTGCTGGACCTAGGCAAGAACCGCATCAAAACGCTCAACCAGGACGAGTTCGCCAGCTTCCCGCACCTGGAGGAGCTGGAGCTCAACGAGAACATCGTGAGCGCCGTGGAGCCCGGCGCCTTCAACAACCTCTTCAACCTCCGGACGCTGGGTCTCCGCAGCAACCGCCTGAAGCTCATCCCGCTAGGCGTCTTCACTGGCCTCAGCAACCTGACCAAGCTGGACATCAGCGAGAACAAGATTGTTATCCTGCTGGACTACATGTTTCAGGACCTGTACAACCTCAAGTCACTGGAGGTTGGCGACAATGACCTCGTCTACATCTCTCACCGCGCCTTCAGCGGCCTCAACAGCCTGGAGCAGCTGACGCTGGAGAAATGCAACCTGACCTCCATCCCCACCGAGGCGCTGTCCCACCTGCACGGCCTCATCGTCCTGAGGCTCCGGCACCTCAACATCAATGCCATCCGGGACTACTCCTTCAAGAGGCTCTACCGACTCAAGGTCTTGGAGATCTCCCACTGGCCCTACTTGGACACCATGACACCCAACTGCCTCTACGGCCTCAACCTGACGTCCCTGTCCATCACACACTGCAATCTGACCGCTGTGCCCTACCTGGCCGTCCGCCACCTAGTCTATCTCCGCTTCCTCAACCTCTCCTACAACCCCATCAGCACCATTGAGGGCTCCATGTTGCATGAGCTGCTCCGGCTGCAGGAGATCCAGCTGGTGGGCGGGCAGCTGGCCGTGGTGGAGCCCTATGCCTTCCGCGGCCTCAACTACCTGCGCGTGCTCAATGTCTCTGGCAACCAGCTGACCACACTGGAGGAATCAGTCTTCCACTCGGTGGGCAACCTGGAGACACTCATCCTGGACTCCAACCCGCTGGCCTGCGACTGTCGGCTCCTGTGGGTGTTCCGGCGCCGCTGGCGGCTCAACTTCAACCGGCAGCAGCCCACGTGCGCCACGCCCGAGTTTGTCCAGGGCAAGGAGTTCAAGGACTTCCCTGATGTGCTACTGCCCAACTACTTCACCTGCCGCCGCGCCCGCATCCGGGACCGCAAGGCCCAGCAGGTGTTTGTGGACGAGGGCCACACGGTGCAGTTTGTGTGCCGGGCCGATGGCGACCCGCCGCCCGCCATCCTCTGGCTCTCACCCCGAAAGCACCTGGTCTCAGCCAAGAGCAATGGGCGGCTCACAGTCTTCCCTGATGGCACGCTGGAGGTGCGCTACGCCCAGGTACAGGACAACGGCACGTACCTGTGCATCGCGGCCAACGCGGGCGGCAACGACTCCATGCCCGCCCACCTGCATGTGCGCAGCTACTCGCCCGACTGGCCCCATCAGCCCAACAAGACCTTCGCTTTCATCTCCAACCAGCCGGGCGAGGGAGAGGCCAACAGCACCCGCGCCACTGTGCCTTTCCCCTTCGACATCAAGACCCTCATCATCGCCACCACCATGGGCTTCATCTCTTTCCTGGGCGTCGTCCTCTTCTGCCTGGTGCTGCTGTTTCTCTGGAGCCGGGGCAAGGGCAACACAAAGCACAACATCGAGATCGAGTATGTGCCCCGAAAGTCGGACGCAGGCATCAGCTCCGCCGACGCGCCCCGCAAGTTCAACATGAAGATGATATGA.

The polypeptide sequence for the full-length LINGO-1 polypeptide is asfollows:

(SEQ ID NO: 51)MLAGGVRSMPSPLLACWQPILLLVLGSVLSGSATGCPPRCECSAQDRAVLCHRKRFVAVPEGIPTETRLLDLGICNRIKTLNQDEFASFPHLEELELNENIVSAVEPGAFNNLFNLRTLGLRSNRLKLIPLGVFTGLSNLTKLDISENKIVILLDYMFQDLYNLKSLEVGDNDLVYISHRAFSGLNSLEQLTLEKCNLTSIPTEALSHLHGLIVLRLRHLNINAIRDYSFKRLYRLKVLEISHWPYLDTMTPNCLYGLNLTSLSITHCNLTAVPYLAVRHLVYLRFLNLSYNPISTIEGSMLHELLRLQEIQLVGGQLAVVEPYAFRGLNYLRVLNVSGNQLTTLEESVFHSVGNLETLILDSNPLACDCRLLWVFRRRWRLNFNRQQPTCATPEFVQGKEFKDFPDVLLPNYFTCRRARIRDRKAQQVFVDEGHTVQFVCRADGDPPPAILWLSPRKHLVSAKSNGRLTVFPDGTLEVRYAQVQDNGTYLCIAANAGGNDSMPAHLHVRSYSPDWPHQPNKTFAFISNQPGEGEANSTRATVPFPFDIKTLIIATTMGFISFLGVVLFCLVLLFLWSRGKGNTKHNIEIEYVPRKSDAGISSADAPRKFNMKMI.

III. Lingo-1 Antibodies

In one embodiment, the present invention is directed to LINGO-1antibodies, or antigen-binding fragments, variants, or derivativesthereof. For example, the present invention includes at least theantigen-binding domains of Li62, Li81 and fragments, variants, andderivatives thereof.

As used herein, the term “antigen binding domain” includes a site thatspecifically binds an epitope on an antigen (e.g., an epitope ofLINGO-1). The antigen binding domain of an antibody typically includesat least a portion of an immunoglobulin heavy chain variable region andat least a portion of an immunoglobulin light chain variable region. Thebinding site formed by these variable regions determines the specificityof the antibody.

The present invention is more specifically directed to a LINGO-1antibody, or antigen-binding fragment, variant or derivatives thereof,where the LINGO-1 antibody binds to the same epitope as Li62 or Li81.

The invention is further drawn to a LINGO-1 antibody, or antigen-bindingfragment, variant or derivatives thereof, where the LINGO-1 antibodycompetitively inhibits Li62 or Li81 from binding to LINGO-1.

The invention is also drawn to a LINGO-1 antibody, or antigen-bindingfragment, variant or derivatives thereof, where the LINGO-1 antibodycomprises at least the antigen binding region of Li62 or Li81.

In certain embodiments, the present invention is directed to anantibody, or antigen-binding fragment, variant, or derivative thereofwhich specifically or preferentially binds to a particular LINGO-1polypeptide fragment or domain. Such LINGO-1 polypeptide fragmentsinclude, but are not limited to, a LINGO-1 polypeptide comprising,consisting essentially of, or consisting of amino acids 34 to 532; 34 to417; 34 to 425; 34 to 493; 66 to 532; 66 to 417; 66 to 426; 66 to 493;66 to 532; 417 to 532; 417 to 425 (the LINGO-1 basic region); 417 to493; 417 to 532; 419 to 493 (the LINGO-1 Ig region); or 425 to 532 ofSEQ ID NO:51; or a LINGO-1 variant polypeptide at least 70%, 75%, 80%,85%, 90%, or 95% identical to amino acids 34 to 532; 34 to 417; 34 to425; 34 to 493; 66 to 532; 66 to 417; 66 to 426; 66 to 493; 66 to 532;417 to 532; 417 to 425 (the LINGO-1 basic region); 417 to 493; 417 to532; 419 to 493 (the LINGO-1 Ig region); or 425 to 532 of SEQ ID NO:51.

Additional LINGO-1 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of one or moreleucine-rich-repeats (LRR) of LINGO-1. Such fragments, include, forexample, fragments comprising, consisting essentially of, or consistingof amino acids 66 to 89; 66 to 113; 66 to 137; 90 to 113; 114 to 137;138 to 161; 162 to 185; 186 to 209; 210 to 233; 234 to 257; 258 to 281;282 to 305; 306 to 329; or 330 to 353 of SEQ ID NO:51. Correspondingfragments of a variant LINGO-1 polypeptide at least 70%, 75%, 80%, 85%,90%, or 95% identical to amino acids 66 to 89; 66 to 113; 90 to 113; 114to 137; 138 to 161; 162 to 185; 186 to 209; 210 to 233; 234 to 257; 258;to 281; 282 to 305; 306 to 329; or 330 to 353 of SEQ ID NO:51 are alsocontemplated.

Additional LINGO-1 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of one or morecysteine rich regions flanking the LRR of LINGO-1. Such fragments,include, for example, a fragment comprising, consisting essentially of,or consisting of amino acids 34 to 64 of SEQ ID NO:51 (the N-terminalLRR flanking region (LRRNT)), or a fragment comprising, consistingessentially of, or consisting of amino acids 363 to 416 of SEQ ID NO:51(the C-terminal LRR flanking region (LRRCT)), amino acids Correspondingfragments of a variant LINGO-1 polypeptide at least 70%, 75%, 80%, 85%,90%, or 95% identical to amino acids 34 to 64 and 363 to 416 of SEQ IDNO:51 are also contemplated.

As known in the art, “sequence identity” between two polypeptides isdetermined by comparing the amino acid sequence of one polypeptide tothe sequence of a second polypeptide. When discussed herein, Whether anyparticular polypeptide is at least about 70%, 75%, 80%, 85%, 90% or 95%identical to another polypeptide can be determined using methods andcomputer programs/software known in the art such as, but not limited to,the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). BESTFIT uses the local homology algorithmof Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981),to find the best segment of homology between two sequences. When usingBESTFIT or any other sequence alignment program to determine whether aparticular sequence is, for example, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference polypeptide sequence and that gaps in homologyof up to 5% of the total number of amino acids in the reference sequenceare allowed.

Additional LINGO-1 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of amino acids 41to 525 of SEQ ID NO:51; 40 to 526 of SEQ ID NO:51; 39 to 527 of SEQ IDNO:51; 38 to 528 of SEQ ID NO:51; 37 to 529 of SEQ ID NO:51; 36 to 530of SEQ ID NO:51; 35 to 531 of SEQ ID NO:51; 34 to 531 of SEQ ID NO:51;46 to 520 of SEQ ID NO:51; 45 to 521 of SEQ ID NO:51; 44 to 522 of SEQID NO:51; 43 to 523 of SEQ ID NO:51; and 42 to 524 of SEQ ID NO:51.

Still additional LINGO-1 peptide fragments to which certain antibodies,or antigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of amino acids 1 to33 of SEQ ID NO:51; 1 to 35 of SEQ ID NO:51; 34 to 64 of SEQ ID NO:51;36 to 64 of SEQ ID NO:51; 66 to 89 of SEQ ID NO: 51; 90 to 113 of SEQ IDNO:51; 114 to 137 of SEQ ID NO:51; 138 to 161 of SEQ ID NO:51; 162 to185 of SEQ ID NO:51; 186 to 209 of SEQ ID NO:51; 210 to 233 of SEQ IDNO:51; 234 to 257 of SEQ ID NO:51; 258 to 281 of SEQ ID NO:51; 282 to305 of SEQ ID NO:51; 306 to 329 of SEQ ID NO:51; 330 to 353 of SEQ IDNO:51; 363 to 416 of SEQ ID NO:51; 417 to 424 of SEQ ID NO:51; 419 to493 of SEQ ID NO:51; and 494 to 551 of SEQ ID NO:51.

Further still, LINGO-1 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of or consisting of amino acids 1 to33 of SEQ ID NO:51; 1 to 35 of SEQ ID NO:51; 1 to 64 of SEQ ID NO:51; to89 of SEQ ID NO:51; 1 to 113 of SEQ ID NO:51; 1 to 137 of SEQ ID NO:51;1 to 161 of SEQ ID NO:51; 1 to 185 of SEQ ID NO:51; 1 to 209 of SEQ IDNO:51; 1 to 233 of SEQ ID NO:51; 1 to 257 of SEQ ID NO:51; 1 to 281 ofSEQ ID NO:51; 1 to 305 of SEQ ID NO:51; 1 to 329 of SEQ ID NO:51; 1 to353 of SEQ ID NO:51; 1 to 416 of SEQ ID NO:51; 1 to 424 of SEQ ID NO:51;1 to 493 of SEQ ID NO:51; 1 to 551 of SEQ ID NO:51; 1 to 531 of SEQ IDNO:51 and 1 to 532 of SEQ ID NO:51.

Additional LINGO-1 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind: include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of amino acids 34to 64 of SEQ ID NO:51; 34 to 89 of SEQ ID NO:51; 34 to 113 of SEQ IDNO:51; 34 to 137 of SEQ ID NO:51; 34 to 161 of SEQ ID NO:51; 34 to 185of SEQ ID NO:51; 34 to 209 of SEQ ID NO:51; 34 to 233 of SEQ ID NO:51;34 to 257 of SEQ ID NO:51; 34 to 281 of SEQ ID NO:51; 34 to 305 of SEQID NO:51; 34 to 329 of SEQ ID NO:51; 34 to 353 of SEQ ID NO:51; 34 to416 of SEQ NO:51; 34 to 424 of SEQ ID NO:51; 34 to 493 of SEQ ID NO:51;and 34 to 551 of SEQ ID NO:51.

More additional LINGO-1 peptide fragments to which certain antibodies,or antigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of or consisting of amino acids 34 to530 of SEQ ID NO:51; 34 to 531 of SEQ ID NO:51; 34 to 532 of SEQ IDNO:51; 34 to 533 of SEQ ID NO:51; 34 to 534 of SEQ ID NO:51; 34 to 535of SEQ ID NO:51; 34 to 536 of SEQ ID NO:51; 34 to 537 of SEQ ID NO:51;34 to 538 of SEQ ID NO:51; 34 to 539 of SEQ ID NO:51; 30 to 532 of SEQNO:51; 31 to 532 of SEQ ID NO:51; 32 to 532 of SEQ ID NO:51; 33 to 532of SEQ ID NO:51; 34 to 532 of SEQ ID NO:51; 35 to 532 of SEQ ID NO:51;36 to 532 of SEQ ID NO:51; 30 to 531 of SEQ ID NO:51; 31 to 531 of SEQID NO:51; 32 to 531 of SEQ ID NO:51; 33 to 531 of SEQ ID NO:51; 34 to531 of SEQ ID NO:51; 35 to 531 of SEQ ID NO:51; and 36 to 531 of SEQ IDNO:51.

Further still, LINGO-1 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of amino acids 36to 64 of SEQ ID NO:51; 36 to 89 of SEQ ID NO:51; 36 to 113 of SEQ IDNO:51; 36 to 137 of SEQ ID NO:51; 36 to 161 of SEQ ID NO:51; 36 to 185of SEQ ID NO:51; 36 to 209 of SEQ ID NO:51; 36 to 233 of SEQ ID NO:51;36 to 257 of SEQ ID NO:51; 36 to 281 of SEQ ID NO:51; 36 to 305 of SEQID NO:51; 36 to 329 of SEQ ID NO:51; 36 to 353 of SEQ ID NO:51; 36 to416 of SEQ ID NO:51; 36 to 424 of SEQ ID NO:51; 36 to 493 of SEQ IDNO:51; and 36 to 551 of SEQ ID NO:51.

Additional LINGO-1 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of amino acids 36to 530 of SEQ ID NO:51; 36 to 531 of SEQ ID NO:51; 36 to 532 of SEQ IDNO:51; 36 to 533 of SEQ ID NO:51; 36 to 534 of SEQ ID NO:51; 36 to 535of SEQ ID NO:51; 36 to 536 of SEQ ID NO:51; 36 to 537 of SEQ ID NO:51;36 to 538 of SEQ ID NO:51; and 36 to 539 of SEQ ID NO:51.

More LINGO-1 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, but are not limited to those fragmentscomprising, consisting essentially of, or consisting of amino acids 417to 493 of SEQ ID NO:51; 417 to 494 of SEQ ID NO:51; 417 to 495 of SEQ IDNO:51; 417 to 496 of SEQ ID NO:51; 417 to 497 of SEQ ID NO:51; 417 to498 of SEQ ID NO:51; 417 to 499 of SEQ ID NO:51; 417 to 500 of SEQ IDNO:51; 417 to 492 of SEQ ID NO:51; 417 to 491 of SEQ ID NO:51; 412 to493 of SEQ ID NO:51; 413 to 493 of SEQ ID NO:51; 414 to 493 of SEQ IDNO:51; 415 to 493 of SEQ ID NO:51; 416 to 493 of SEQ ID NO:51; 411 to493 of SEQ ID NO:51; 410 to 493 of SEQ ID NO:51; 410 to 494 of SEQ IDNO:51; 411 to 494 of SEQ ID NO:51; 412 to 494 of SEQ ID NO:51; 413 to494 of SEQ ID NO:51; 414 to 494 of SEQ ID NO:51; 415 to 494 of SEQ IDNO:51; 416 to 494 of SEQ ID NO:51; 417 to 494 of SEQ ID NO:51; and 418to 494 of SEQ ID NO:51.

In an additional embodiment LINGO-1 peptide fragments to which certainantibodies, or antigen-binding fragments, variants, or derivativesthereof of the present invention bind include, a LINGO-1 polypeptidecomprising, consisting essentially of, or consisting of peptides of theIg domain of LINGO-1 or fragments, variants, or derivatives of suchpolypeptides. Specifically, polypeptides comprising, consistingessentially of, or consisting of the following polypeptide sequences:ITX₁X₂X₃ (SEQ ID NO:88), ACX₁X₂X₃ (SEQ ID NO:89), VCX₁X₂X₃ (SEQ IDNO:90) and SPX₁X₂X₃ (SEQ ID NO:91) where X₁ is lysine, arginine,histidine, glutamine, or asparagine, X₂ is lysine, arginine, histidine,glutamine, or asparagine and X₃ is lysine, arginine, histidine,glutamine, or asparagine. For example, LINGO-1 peptide fragments towhich certain antibodies, or antigen-binding fragments, variants, orderivatives thereof of the present invention bind include, thosefragments comprising, consisting essentially of, or consisting of thefollowing polypeptide sequences: SPRKH (SEQ ID NO:92), SPRKK (SEQ IDNO:93), SPRKR (SEQ ID NO:94), SPKKH (SEQ ID NO:95), SPHKH (SEQ IDNO:96), SPRRH (SEQ ID NO:97), SPRHH (SEQ ID NO:98), SPRRR (SEQ IDNO:99), SPHHH (SEQ ID NO:100) SPKKK (SEQ ID NO:101), LSPRKH (SEQ IDNO:102), LSPRKK (SEQ ID NO:103), LSPRKR (SEQ ID NO:104), LSPKKH (SEQ IDNO:105), LSPHKH (SEQ ID NO:106), LSPRRH (SEQ ID NO:107), LSPRHH (SEQ IDNO:108), LSPRRR (SEQ ID NO:109), LSPHHH (SEQ ID NO:110) LSPKKK (SEQ IDNO:111), WLSPRKH (SEQ ID NO:112), WLSPRKK (SEQ ID NO:113), WLSPRKR (SEQID NO:114), WLSPKKH (SEQ ID NO:115), WLSPHKH (SEQ ID NO:116), WLSPRRH(SEQ ID NO:117), WLSPRHH (SEQ ID NO:118), WLSPRRR (SEQ ID NO:119),WLSPHHH (SEQ ID NO:120) WLSPKKK (SEQ ID NO:121). These LINGO-1polypeptides include the basic “RKH loop” (Arginine-Lysine-Histidineamino acids 456-458) in the Ig domain of LINGO-1. Additional LINGO-1peptides which include a basic tripeptide are ITPKRR (SEQ ID NO:122),ACHHK (SEQ ID NO:123) and VCHHK (SEQ ID NO:124).

Additional LINGO-1 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, a LINGO-1 polypeptide comprising,consisting essentially of, or consisting of peptides of the Ig domain ofLINGO-1 or fragments, variants, or derivatives of such polypeptides.Specifically, peptides comprising, consisting essentially of, orconsisting of the following polypeptide sequences: X₄X₅RKH (SEQ IDNO:125), X₄X₅RRR (SEQ ID NO:126), X₄X₅KKK (SEQ ID NO:127), X₄X₅HHH (SEQID NO:128), X₄X₅RKK (SEQ ID NO:129), X₄X₅RKR (SEQ ID NO:130), X₄X₅KKH(SEQ ID NO:131), X₄X₅HKH (SEQ ID NO:132), X₄X₅RRH (SEQ ID NO:133) andX₄X₅RHH (SEQ ID NO:134) where X₄ is any amino acid and X₅ is any aminoacid.

In other embodiments LINGO-1 peptide fragments to which certainantibodies, or antigen-binding fragments, variants, or derivativesthereof of the present invention bind include, a LINGO-1 polypeptidecomprising, consisting essentially of, or consisting of peptides of theIg domain of LINGO-1 or fragments, variants, or derivatives of suchpolypeptides. Specifically, polypeptides comprising, consistingessentially of, or consisting of the following polypeptide sequences:ITX₆X₇X₈ (SEQ ID NO:135), ACX₆X₇X₈ (SEQ ID NO:136), VCX₆X₇X₈ (SEQ IDNO:137) and SPX₆X₇X₈ (SEQ ID NO:138) where X₆ is lysine, arginine,histidine, glutamine, or asparagine, X₇ is any amino acid and X₈ islysine, arginine, histidine, glutamine, or asparagine. For example, apolypeptide comprising, consisting essentially of, or consisting of thefollowing polypeptide sequence: SPRLH (SEQ ID NO:139).

LINGO-1 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, a LINGO-1 polypeptide comprising,consisting essentially of, or consisting of peptides which contain aminoacids 452-458 in the Ig domain of LINGO-1, or derivatives thereof,wherein amino acid 452 is a tryptophan or phenylalanine residue.

Additional LINGO-1 peptide fragments to which certain antibodies, orantigen-binding fragments, variants, or derivatives thereof of thepresent invention bind include, a LINGO-1 polypeptide comprising,consisting essentially of, or consisting of peptides of the basic domainof LINGO-1. Specifically, peptides comprising, consisting essentiallyof, or consisting of the following polypeptide sequences: RRARIRDRK (SEQID NO:140), KKVKVKEKR (SEQ ID NO:141), RRLRLRDRK (SEQ ID NO:142),RRGRGRDRK (SEQ ID NO:143) and RRIRARDRK (SEQ ID NO:144).

Additional exemplary soluble LINGO-1 polypeptides and methods andmaterials for obtaining these molecules for producing antibodies orantibody fragments of the present invention may be found, e.g., inInternational Patent Application No. PCT/US2004/008323, incorporatedherein by reference in its entirety.

Methods of making antibodies are well known in the art and describedherein. Once antibodies to various fragments of, or to the full-lengthLINGO-1 without the signal sequence, have been produced, determiningwhich amino acids, or epitope, of LINGO-1 to which the antibody orantigen binding fragment binds can be determined by eptiope mappingprotocols as described herein as well as methods known in the art (e.g.double antibody-sandwich ELISA as described in “Chapter 11—Immunology,”Current Protocols in Molecular Biology, Ed. Ausubel et al., v.2, JohnWiley & Sons, Inc. (1996)). Additional epitope mapping protocols may befound in Morris, G. Epitope Mapping Protocols, New Jersey: Humana Press(1996), which are both incorporated herein by reference in theirentireties. Epitope mapping can also be performed by commerciallyavailable means (i.e. ProtoPROBE, Inc. (Milwaukee, Wis.)).

Additionally, antibodies produced which bind to any portion of LINGO-1can then be screened for their ability to act as an antagonist ofLINGO-1 and thus promote neurite outgrowth, neuronal and oligodendrocytesurvival, proliferation and differentiation as well as promotemyelination. Antibodies can be screened for oligodendrocyte/neuronalsurvival for example by using the methods described herein such as inExamples 11 or 12 or as described in PCT/US2008/000316, filed Jan. 9,2008, and PCT/US2006/026271, filed Jul. 7, 2006, which are incorporatedherein by reference in their entireties. Additionally, antibodies can bescreened for example by their ability to promote myelination by usingthe methods described herein such as in Examples 2, 6, 9, 10, 11 or 13or as described in PCT/US2008/000316 and/or PCT/US2006/026271. Finally,antibodies can be screened for their ability to promote oligodendrocyteproliferation and differentiation, as well as neurite outgrowth forexample by using the methods described herein such as in Examples 4 or 5or as described in PCT/US2008/000316 and/or PCT/US2006/026271. Otherantagonist functions of antibodies of the present invention can betested using other assays as described in the Examples herein.

In other embodiments, the present invention includes an antibody, orantigen-binding fragment, variant, or derivative thereof whichspecifically or preferentially binds to at least one epitope of LINGO-1,where the epitope comprises, consists essentially of, or consists of atleast about four to five amino acids of SEQ ID NO:51, at least seven, atleast nine, or between at least about 15 to about 30 amino acids of SEQID NO:51. The amino acids of a given epitope of SEQ ID NO:51 asdescribed may be, but need not be contiguous or linear. In certainembodiments, the at least one epitope of LINGO-1 comprises, consistsessentially of, or consists of a non-linear epitope formed by theextracellular domain of LINGO-1 as expressed on the surface of a cell oras a soluble fragment, e.g., fused to an IgG Fc region. Thus, in certainembodiments the at least one epitope of LINGO-1 comprises, consistsessentially of, or consists of at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 15, at least 20,at least 25, between about 15 to about 30, or at least 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100contiguous or non-contiguous amino acids of SEQ ID NO:51, where thenon-contiguous amino acids form an epitope through protein folding.

In other embodiments, the present invention includes an antibody, orantigen-binding fragment, variant, or derivative thereof whichspecifically or preferentially binds to at least one epitope of LINGO-1,where the epitope comprises, consists essentially of, or consists of, inaddition to one, two, three, four, five, six or more contiguous ornon-contiguous amino acids of SEQ ID NO:51 as described above, and anadditional moiety which modifies the protein, e.g., a carbohydratemoiety may be included such that the LINGO-1 antibody binds with higheraffinity to modified target protein than it does to an unmodifiedversion of the protein. Alternatively, the LINGO-1 antibody does notbind the unmodified version of the target protein at all.

In certain aspects, the present invention is directed to an antibody, orantigen-binding fragment, variant, or derivative thereof whichspecifically binds to a LINGO-1 polypeptide or fragment thereof, or aLINGO-1 variant polypeptide, with an affinity characterized by adissociation constant (K_(D)) which is less than the K_(D) for saidreference monoclonal antibody.

In certain embodiments, an antibody, or antigen-binding fragment,variant, or derivative thereof of the invention binds specifically to atleast one epitope of LINGO-1 or fragment or variant described above,i.e., binds to such an epitope more readily than it would bind to anunrelated, or random epitope; binds preferentially to at least oneepitope of LINGO-1 or fragment or variant described above, i.e., bindsto such an epitope more readily than it would bind to a related,similar, homologous, or analogous epitope; competitively inhibitsbinding of a reference antibody which itself binds specifically orpreferentially to a certain epitope of LINGO-1 or fragment or variantdescribed above; or binds to at least one epitope of LINGO-1 or fragmentor variant described above with an affinity characterized by adissociation constant K_(D) of less than about 5×10⁻² M, about 10⁻² M,about 5×10⁻³M, about 10⁻³M, about 5×10⁻⁴M, about 10⁻⁴M, about 5×10⁻⁵M,about 10⁻⁵M, about 5×10⁻⁶M, about 10⁻⁶M, about 5×10⁻⁷M, about 10⁻⁷M,about 5×10⁻⁸M, about 10⁻⁸M, about 5×10⁻⁹M, about 10⁻⁹M, about 5×10⁻¹⁰ M,about 10⁻¹⁰ M, about 5×10⁻¹¹M, about 10⁻¹¹M, about 5×10⁻¹² M, about10⁻¹² M, about 5×10⁻¹³M, about 10⁻¹³ M, about 5×10⁻¹⁴ M, about 10⁻¹⁴ M,about 5×10⁻¹⁵ M, or about 10⁻¹⁵ M. In a particular aspect, the antibodyor fragment thereof preferentially binds to a humanLINGO-1 polypeptideor fragment thereof, relative to a murine LINGO-1 polypeptide orfragment thereof.

As used in the context of antibody binding dissociation constants, theterm “about” allows for the degree of variation inherent in the methodsutilized for measuring antibody affinity. For example, depending on thelevel of precision of the instrumentation used, standard error based onthe number of samples measured, and rounding error, the term “about10⁻²M” might include, for example, from 0.05 M to 0.005 M.

In specific embodiments, an antibody, or antigen-binding fragment,variant, or derivative thereof of the invention binds LINGO-1polypeptides or fragments or variants thereof with an off rate (k(off))of less than or equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹ or 10⁻³sec⁻¹. Alternatively, an antibody, or antigen-binding fragment, variant,or derivative thereof of the invention binds binds LINGO-1 polypeptidesor fragments or variants thereof with an off rate (k(off)) of less thanor equal to 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹ 5×10⁻⁶sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷ sec⁻¹.

In other embodiments, an antibody, or antigen-binding fragment, variant,or derivative thereof of the invention binds LINGO-1 polypeptides orfragments or variants thereof with an on rate (k(on)) of greater than orequal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹ or 5×10⁴ M⁻¹sec⁻. Alternatively, an antibody, or antigen-binding fragment, variant,or derivative thereof of the invention binds LINGO-1 polypeptides orfragments or variants thereof with an on rate (k(on)) greater than orequal to 10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×106 M⁻¹sec⁻¹ or 10⁷ M⁻¹ sec⁻¹.

In various embodiments, a LINGO-1 antibody, or antigen-binding fragment,variant, or derivative thereof as described herein is an antagonist ofLINGO-1 activity. In certain embodiments, for example, binding of anantagonist LINGO-1 antibody to LINGO-1, as expressed on neurons, blocksmyelin-associated neurite outgrowth inhibition or neuronal cell death.In other embodiments, binding of the LINGO-1 antibody to LINGO-1, asexpressed on oligodendrocytes, blocks inhibition of oligodendrocytegrowth or differentiation, or blocks demyelination or dysmyelination ofCNS neurons.

Unless it is specifically noted, as used herein a “fragment thereof” inreference to an antibody refers to an antigen-binding fragment, i.e., aportion of the antibody which specifically binds to the antigen. In oneembodiment, a LINGO-1 antibody, e.g., an antibody of the invention is abispecific LINGO-1 antibody, binding polypeptide, or antibody, e.g., abispecific antibody, minibody, domain deleted antibody, or fusionprotein having binding specificity for more than one epitope, e.g., morethan one antigen or more than one epitope on the same antigen. In oneembodiment, a bispecific LINGO-1 antibody, binding polypeptide, orantibody has at least one binding domain specific for at least oneepitope on a target polypeptide disclosed herein, e.g., LINGO-1. Inanother embodiment, a bispecific LINGO-1 antibody, binding polypeptide,or antibody has at least one binding domain specific for an epitope on atarget polypeptide and at least one target binding domain specific for adrug or toxin. In yet another embodiment, a bispecific LINGO-1 antibody,binding polypeptide, or antibody has at least one binding domainspecific for an epitope on a target polypeptide disclosed herein, and atleast one binding domain specific for a prodrug. A bispecific LINGO-1antibody, binding polypeptide, or antibody may be a tetravalent antibodythat has two target binding domains specific for an epitope of a targetpolypeptide disclosed herein and two target binding domains specific fora second target. Thus, a tetravalent bispecific LINGO-1 antibody,binding polypeptide, or antibody may be bivalent for each specificity.

LINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention, as known by those of ordinaryskill in the art, can comprise a constant region which mediates one ormore effector functions. For example, binding of the C1 component ofcomplement to an antibody constant region may activate the complementsystem. Activation of complement is important in the opsonisation andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and may also be involved in autoimmunehypersensitivity. Further, antibodies bind to receptors on various cellsvia the Fc region, with a Fc receptor binding site on the antibody Fcregion binding to a Fc receptor (FcR) on a cell. There are a number ofFc receptors which are specific for different classes of antibody,including IgG (gamma receptors), IgE (epsilon receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of antibody to Fc receptorson cell surfaces triggers a number of important and diverse biologicalresponses including engulfment and destruction of antibody-coatedparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (called antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer and control of immunoglobulin production.

Accordingly, certain embodiments of the invention include a LINGO-1antibody, or antigen-binding fragment, variant, or derivative thereof,in which at least a fraction of one or more of the constant regiondomains has been deleted or otherwise altered so as to provide desiredbiochemical characteristics such as reduced effector functions, theability to non-covalently dimerize, increased ability to localize at thesite of a tumor, reduced serum half-life, or increased serum half-lifewhen compared with a whole, unaltered antibody of approximately the sameimmunogenicity. For example, certain antibodies for use in thediagnostic and treatment methods described herein are domain deletedantibodies which comprise a polypeptide chain similar to animmunoglobulin heavy chain, but which lack at least a portion of one ormore heavy chain domains. For instance, in certain antibodies, oneentire domain of the constant region of the modified antibody will bedeleted, for example, all or part of the C_(H)2 domain will be deleted.

In certain LINGO-1 antibodies, or antigen-binding fragments, variants,or derivatives thereof described herein, the Fc portion may be mutatedto decrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce Fc receptor binding of thecirculating modified antibody thereby increasing tumor localization. Inother cases it may be that constant region modifications consistent withthe instant invention moderate complement binding and thus reduce theserum half life and nonspecific association of a conjugated cytotoxin.Yet other modifications of the constant region may be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. The resulting physiological profile, bioavailability andother biochemical effects of the modifications, such as tumorlocalization, biodistribution and serum half-life, may easily bemeasured and quantified using well know immunological techniques withoutundue experimentation.

Modified forms of LINGO-1 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention can be made from wholeprecursor or parent antibodies using techniques known in the art.Exemplary techniques are discussed in more detail herein.

In certain embodiments both the variable and constant regions of LINGO-1antibodies, or antigen-binding fragments, variants, or derivativesthereof are fully human. Fully human antibodies can be made usingtechniques that are known in the art and as described herein. Forexample, fully human antibodies against a specific antigen can beprepared by administering the antigen to a transgenic animal which hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled. Exemplarytechniques that can be used to make such antibodies are described inU.S. Pat. Nos. 6,150,584; 6,458,592; 6,420,140 which are incorporated byreference in their entireties. Other techniques are known in the art.Fully human antibodies can likewise be produced by various displaytechnologies, e.g., phage display or other viral display systems, asdescribed in more detail elsewhere herein.

LINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be made or manufactured usingtechniques that are known in the art. In certain embodiments, antibodymolecules or fragments thereof are “recombinantly produced,” i.e., areproduced using recombinant DNA technology. Exemplary techniques formaking antibody molecules or fragments thereof are discussed in moredetail elsewhere herein.

LINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention also include derivatives that aremodified, e.g., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom specifically binding to its cognate epitope. For example, but notby way of limitation, the antibody derivatives include antibodies thathave been modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

In certain embodiments, LINGO-1 antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention will notelicit a deleterious immune response in the animal to be treated, e.g.,in a human. In one embodiment, LINGO-1 antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention aremodified to reduce their immunogenicity using art-recognized techniques.For example, antibodies can be humanized, primatized, deimmunized, orchimeric antibodies can be made. These types of antibodies are derivedfrom a non-human antibody, typically a murine or primate antibody, thatretains or substantially retains the antigen-binding properties of theparent antibody, but which is less immunogenic in humans. This may beachieved by various methods, including (a) grafting the entire non-humanvariable domains onto human constant regions to generate chimericantibodies; (b) grafting at least a part of one or more of the non-humancomplementarity determining regions (CDRs) into a human framework andconstant regions with or without retention of critical frameworkresidues; or (c) transplanting the entire non-human variable domains,but “cloaking” them with a human-like section by replacement of surfaceresidues. Such methods are disclosed in Morrison et al., Proc. Natl.Acad. Sci. 81:6851-6855 (1984); Morrison et al., Adv. Immunol. 44:65-92(1988); Verhoeyen et al., Science 239:1534-1536 (1988); Padlan, Molec.Immun. 28:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994), andU.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and 6,190,370, all ofwhich are hereby incorporated by reference in their entirety.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” includes alterationof an antibody to modify T cell epitopes (see, e.g., WO9852976A1,WO0034317A2). For example, V_(H) and V_(L) sequences from the startingantibody are analyzed and a human T cell epitope “map” from each Vregion showing the location of epitopes in relation tocomplementarity-determining regions (CDRs) and other key residues withinthe sequence. Individual T cell epitopes from the T cell epitope map areanalyzed in order to identify alternative amino acid substitutions witha low risk of altering activity of the final antibody. A range ofalternative V_(H) and V_(L) sequences are designed comprisingcombinations of amino acid substitutions and these sequences aresubsequently incorporated into a range of binding polypeptides, e.g.,LINGO-1-specific antibodies or immunospecific fragments thereof for usein the diagnostic and treatment methods disclosed herein, which are thentested for function. Typically, between 12 and 24 variant antibodies aregenerated and tested. Complete heavy and light chain genes comprisingmodified V and human C regions are then cloned into expression vectorsand the subsequent plasmids introduced into cell lines for theproduction of whole antibody. The antibodies are then compared inappropriate biochemical and biological assays, and the optimal variantis identified.

LINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen of interestcan be produced by various procedures well known in the art. Forexample, a LINGO-1 antibody, e.g., a binding polypeptide, e.g., aLINGO-1-specific antibody or immunospecific fragment thereof can beadministered to various host animals including, but not limited to,rabbits, mice, rats, chickens, hamsters, goats, donkeys, etc., to inducethe production of sera containing polyclonal antibodies specific for theantigen. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, and include but are not limitedto, Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Suchadjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.(1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas Elsevier, N.Y., 563-681 (1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced. Thus, the term“monoclonal antibody” is not limited to antibodies produced throughhybridoma technology. Monoclonal antibodies can be prepared usingLINGO-1 knockout mice to increase the regions of epitope recognition.Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma and recombinant andphage display technology as described elsewhere herein.

Using art recognized protocols, in one example, antibodies are raised inmammals by multiple subcutaneous or intraperitoneal injections of therelevant antigen (e.g., purified tumor associated antigens such asLINGO-1 or cells or cellular extracts comprising such antigens) and anadjuvant. This immunization typically elicits an immune response thatcomprises production of antigen-reactive antibodies from activatedsplenocytes or lymphocytes. While the resulting antibodies may beharvested from the serum of the animal to provide polyclonalpreparations, it is often desirable to isolate individual lymphocytesfrom the spleen, lymph nodes or peripheral blood to provide homogenouspreparations of monoclonal antibodies (MAbs). Preferably, thelymphocytes are obtained from the spleen.

In this well known process (Kohler et al., Nature 256:495 (1975)) therelatively short-lived, or mortal, lymphocytes from a mammal which hasbeen injected with antigen are fused with an immortal tumor cell line(e.g. a myeloma cell line), thus, producing hybrid cells or “hybridomas”which are both immortal and capable of producing the genetically codedantibody of the B cell. The resulting hybrids are segregated into singlegenetic strains by selection, dilution, and regrowth with eachindividual strain comprising specific genes for the formation of asingle antibody. They produce antibodies which are homogeneous against adesired antigen and, in reference to their pure genetic parentage, aretermed “monoclonal.”

Hybridoma cells thus prepared are seeded and grown in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. Those skilledin the art will appreciate that reagents, cell lines and media for theformation, selection and growth of hybridomas are commercially availablefrom a number of sources and standardized protocols are wellestablished. Generally, culture medium in which the hybridoma cells aregrowing is assayed for production of monoclonal antibodies against thedesired antigen. Preferably, the binding specificity of the monoclonalantibodies produced by hybridoma cells is determined by in vitro assayssuch as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). After hybridoma cells are identified thatproduce antibodies of the desired specificity, affinity and/or activity,the clones may be subcloned by limiting dilution procedures and grown bystandard methods (Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, pp 59-103 (1986)). It will further beappreciated that the monoclonal antibodies secreted by the subclones maybe separated from culture medium, ascites fluid or serum by conventionalpurification procedures such as, for example, protein-A, hydroxylapatitechromatography, gel electrophoresis, dialysis or affinitychromatography.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments may be producedby proteolytic cleavage of immunoglobulin molecules, using enzymes suchas papain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). F(ab′)₂ fragments contain the variable region, the lightchain constant region and the C_(H)1 domain of the heavy chain.

Those skilled in the art will also appreciate that DNA encodingantibodies or antibody fragments (e.g., antigen binding sites) may alsobe derived from antibody libraries, such as phage display libraries. Ina particular, such phage can be utilized to display antigen-bindingdomains expressed from a repertoire or combinatorial antibody library(e.g., human or murine). Phage expressing an antigen binding domain thatbinds the antigen of interest can be selected or identified withantigen, e.g., using labeled antigen or antigen bound or captured to asolid surface or bead. Phage used in these methods are typicallyfilamentous phage including fd and M13 binding domains expressed fromphage with Fab, Fv OE DAB (individival Fv region from light or heavychains) or disulfide stabilized Fv antibody domains recombinantly fusedto either the phage gene III or gene VIII protein. Exemplary methods areset forth, for example, in EP 368 684 B1; U.S. Pat. No. 5,969,108,Hoogenboom, H. R. and Chames, Immunol. Today 21:371 (2000); Nagy et al.Nat. Med. 8:801 (2002); Huie et al., Proc. Natl. Acad. Sci. USA 98:2682(2001); Lui et al., J. Mol. Biol. 315:1063 (2002), each of which isincorporated herein by reference. Several publications (e.g., Marks etal., Bio/Technology 10:779-783 (1992)) have described the production ofhigh affinity human antibodies by chain shuffling, as well ascombinatorial infection and in vivo recombination as a strategy forconstructing large phage libraries. In another embodiment, Ribosomaldisplay can be used to replace bacteriophage as the display platform(see, e.g., Hanes et al., Nat. Biotechnol. 18:1287 (2000); Wilson etal., Proc. Natl. Acad. Sci. USA 98:3750 (2001); or Irving et al., J.Immunol. Methods 248:31 (2001)). In yet another embodiment, cell surfacelibraries can be screened for antibodies (Boder et al., Proc. Natl.Acad. Sci. USA 97:10701 (2000); Daugherty et al., J. Immunol. Methods243:211 (2000)). Such procedures provide alternatives to traditionalhybridoma techniques for the isolation and subsequent cloning ofmonoclonal antibodies.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. For example, DNA sequences encoding V_(H) and V_(L)regions are amplified from animal cDNA libraries (e.g., human or murinecDNA libraries of lymphoid tissues) or synthetic cDNA libraries. Incertain embodiments, the DNA encoding the V_(H) and V_(L) regions arejoined together by an scFv linker by PCR and cloned into a phagemidvector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is electroporatedin E. coli and the E. coli is infected with helper phage. Phage used inthese methods are typically filamentous phage including fd and M13 andthe V_(H) or V_(L) regions are usually recombinantly fused to either thephage gene III or gene VIII. Phage expressing an antigen binding; domainthat binds to an antigen of interest (i.e., a LINGO-1 polypeptide or afragment thereof) can be selected or identified with antigen, e.g.,using labeled antigen or antigen bound or captured to a solid surface orhead.

Additional examples of phage display methods that can be used to makethe antibodies include those disclosed in Brinkman et al., J. Immunol.Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186(1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persicet al., Gene 187:9-18 (1997); Burton et al., Advances in Immunology57:191-280 (1994); PCT Application No. PCT/GB91/01134; PCT publicationsWO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′ andF(ab′)₂ fragments can also be employed using methods known in the artsuch as those disclosed in PCT publication WO 92/22324; Mullinax et al.,BioTechniques 12(6):864-869 (1992); and Sawai et al., AJR134:26-34(1995); and Better et al., Science 240:1041-1043 (1988) (said referencesincorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporatedherein by reference in their entireties. Humanized antibodies areantibody molecules derived from a non-human species antibody that bindthe desired antigen having one or more complementarity determiningregions (CDRs) from the non-human species and framework regions from ahuman immunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332, which is incorporated by reference in its entirety).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a desired target polypeptide. Monoclonal antibodies directedagainst the antigen can be obtained from the immunized, transgenic miceusing conventional hybridoma technology. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B-celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA, IgM and IgE antibodies. For an overviewof this technology for producing human antibodies, see Lonberg andHuszar Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion ofthis technology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; and 5,939,598, which are incorporated by reference herein intheir entirety. In addition, companies such as Abgenix, Inc. (Freemont,Calif.) and GenPharm (San Jose, Calif.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/Technology 12:899-903(1988). See also, U.S. Pat. No. 5,565,332, which is incorporated byreference in its entirety.)

Further, antibodies to target polypeptides of the invention can, inturn, be utilized to generate anti-idiotype antibodies that “mimic”target polypeptides using techniques well known to those skilled in theart. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444 (1989) andNissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodieswhich bind to and competitively inhibit polypeptide multimerizationand/or binding of a polypeptide of the invention to a ligand can be usedto generate anti-idiotypes that “mimic” the polypeptide multimerizationand/or binding domain and, as a consequence, bind to and neutralizepolypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fabfragments of such anti-idiotypes can be used in therapeutic regimens toneutralize polypeptide ligand. For example, such anti-idiotypicantibodies can be used to bind a desired target polypeptide and/or tobind its ligands/receptors, and thereby block its biological activity.

In another embodiment, DNA encoding desired monoclonal antibodies may bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Theisolated and subcloned hybridoma cells serve as a preferred source ofsuch DNA. Once isolated, the DNA may be placed into expression vectors,which are then transfected into prokaryotic or eukaryotic host cellssuch as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO)cells or myeloma cells that do not otherwise produce immunoglobulins.More particularly, the isolated DNA (which may be synthetic as describedherein) may be used to clone constant and variable region sequences forthe manufacture antibodies as described in Newman et al., U.S. Pat. No.5,658,570, filed Jan. 25, 1995, which is incorporated by referenceherein. Essentially, this entails extraction of RNA from the selectedcells, conversion to cDNA, and amplification by PCR using Ig specificprimers. Suitable primers for this purpose are also described in U.S.Pat. No. 5,658,570. As will be discussed in more detail below,transformed cells expressing the desired antibody may be grown up inrelatively large quantities to provide clinical and commercial suppliesof the immunoglobulin.

In one embodiment, a LINGO-1 antibody of the invention comprises atleast one heavy or light chain CDR of an antibody molecule. In anotherembodiment, a LINGO-1 antibody of the invention comprises at least twoCDRs from one or more antibody molecules. In another embodiment, aLINGO-1 antibody of the invention comprises at least three CDRs from oneor more antibody molecules. In another embodiment, a LINGO-1 antibody ofthe invention comprises at least four CDRs from one or more antibodymolecules. In another embodiment, a LINGO-1 antibody of the inventioncomprises at least five CDRs from one or more antibody molecules. Inanother embodiment, a LINGO-1 antibody of the invention comprises atleast six CDRs from one or more antibody molecules. Exemplary antibodymolecules comprising at least one CDR that can be included in thesubject LINGO-1 antibodies are described herein.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody. Theframework regions may be naturally occurring or consensus frameworkregions, and preferably human framework regions (see, e.g., Chothia etal., J. Mol. Biol. 278:457-479 (1998) for a listing of human frameworkregions). Preferably, the polynucleotide generated by the combination ofthe framework regions and CDRs encodes an antibody that specificallybinds to at least one epitope of a desired polypeptide, e.g., LINGO-1.Preferably, one or more amino acid substitutions may be made within theframework regions, and, preferably, the amino acid substitutions improvebinding of the antibody to its antigen. Additionally, such methods maybe used to make amino acid substitutions or deletions of one or morevariable region cysteine residues participating in an intrachaindisulfide bond to generate antibody molecules lacking one or moreintrachain disulfide bonds. Other alterations to the polynucleotide areencompassed by the present invention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asused herein, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,694,778; Bird, Science 242:423-442 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-554 (1989)) can be adapted to produce singlechain antibodies. Single chain antibodies are formed by linking theheavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain antibody. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science 242:1038-1041 (1988)).

Yet other embodiments of the present invention comprise the generationof human or substantially human antibodies in transgenic animals (e.g.,mice) that are incapable of endogenous immunoglobulin production (seee.g., U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591,669 and 5,589,369 eachof which is incorporated herein by reference). For example, it has beendescribed that the homozygous deletion of the antibody heavy-chainjoining region in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of a humanimmunoglobulin gene array to such germ line mutant mice will result inthe production of human antibodies upon antigen challenge. Anotherpreferred means of generating human antibodies using SCID mice isdisclosed in U.S. Pat. No. 5,811,524 which is incorporated herein byreference. It will be appreciated that the genetic material associatedwith these human antibodies may also be isolated and manipulated asdescribed herein.

Yet another highly efficient means for generating recombinant antibodiesis disclosed by Newman, Biotechnology 10: 1455-1460 (1992).Specifically, this technique results in the generation of primatizedantibodies that contain monkey variable domains and human constantsequences. This reference is incorporated by reference in its entiretyherein. Moreover, this technique is also described in commonly assignedU.S. Pat. Nos. 5,658,570, 5,693,780 and 5,756,096 each of which isincorporated herein by reference.

In another embodiment, lymphocytes can be selected by micromanipulationand the variable genes isolated. For example, peripheral bloodmononuclear cells can be isolated from an immunized mammal and culturedfor about 7 days in vitro. The cultures can be screened for specificIgGs that meet the screening criteria. Cells from positive wells can beisolated. Individual Ig-producing B cells can be isolated by FACS or byidentifying them in a complement-mediated hemolytic plaque assay.Ig-producing B cells can be micromanipulated into a tube and the V_(H)and V_(L) genes can be amplified using, e.g., RT-PCR. The V_(H) andV_(L) genes can be cloned into an antibody expression vector andtransfected into cells (e.g., eukaryotic or prokaryotic cells) forexpression.

Alternatively, antibody-producing cell lines may be selected andcultured using techniques well known to the skilled artisan. Suchtechniques are described in a variety of laboratory manuals and primarypublications. In this respect, techniques suitable for use in theinvention as described below are described in Current Protocols inImmunology, Coligan et al., Eds., Green Publishing Associates andWiley-Interscience, John Wiley and Sons, New York (1991) which is hereinincorporated by reference in its entirety, including supplements.

Antibodies for use in the diagnostic and therapeutic methods disclosedherein can be produced by any method known in the art for the synthesisof antibodies, in particular, by chemical synthesis or preferably, byrecombinant expression techniques as described herein.

In one embodiment, a LINGO-1 antibody, or antigen-binding fragment,variant, or derivative thereof of the invention comprises a syntheticconstant region wherein one or more domains are partially or entirelydeleted (“domain-deleted antibodies”). In certain embodiments compatiblemodified antibodies will comprise domain deleted constructs or variantswherein the entire C_(H)2 domain has been removed (ΔC_(H)2 constructs).For other embodiments a short connecting peptide may be substituted forthe deleted domain to provide flexibility and freedom of movement forthe variable region. Those skilled in the art will appreciate that suchconstructs are particularly preferred due to the regulatory propertiesof the C_(H)2 domain on the catabolic rate of the antibody. Domaindeleted constructs can be derived using a vector (e.g., from Biogen IDECIncorporated) encoding an IgG1 human constant domain (see, e.g., WO02/060955A2 and WO02/096948A2, which are incorporated by reference intheir entireties). This exemplary vector was engineered to delete theC_(H)2 domain and provide a synthetic vector expressing a domain deletedIgG1 constant region.

In certain embodiments, LINGO-1 antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention areminibodies. Minibodies can be made using methods described in the art(see, e.g., see e.g., U.S. Pat. No. 5,837,821 or WO 94/09817A1, whichare incorporated by reference in their entireties).

In one embodiment, a LINGO-1 antibody, or antigen-binding fragment,variant, or derivative thereof of the invention comprises animmunoglobin heavy chain having deletion or substitution of a few oreven a single amino acid as long as it permits association between themonomeric subunits. For example, the mutation of a single amino acid inselected areas of the C_(H)2 domain may be enough to substantiallyreduce Fc binding and thereby increase tumor localization. Similarly, itmay be desirable to simply delete that part of one or more constantregion domains that control the effector function (e.g. complementbinding) to be modulated. Such partial deletions of the constant regionsmay improve selected characteristics of the antibody (serum half-life)while leaving other desirable functions associated with the subjectconstant region domain intact. Moreover, as alluded to above, theconstant regions of the disclosed antibodies may be synthetic throughthe mutation or substitution of one or more amino acids that enhancesthe profile of the resulting construct. In this respect it may bepossible to disrupt the activity provided by a conserved binding site(e.g. Fc binding) while substantially maintaining the configuration andimmunogenic profile of the modified antibody. Yet other embodimentscomprise the addition of one or more amino acids to the constant regionto enhance desirable characteristics such as effector function orprovide for more cytotoxin or carbohydrate attachment. In suchembodiments it may be desirable to insert or replicate specificsequences derived from selected constant region domains.

The present invention also provides antibodies that comprise, consistessentially of, or consist of, variants (including derivatives) ofantibody molecules (e.g., the V_(H) regions and/or V_(L) regions)described herein, which antibodies or fragments thereofimmunospecifically bind to a LINGO-1 polypeptide or fragment or variantthereof. Standard techniques known to those of skill in the art can beused to introduce mutations in the nucleotide sequence encoding aLINGO-1 antibody, including, but not limited to, site-directedmutagenesis and PCR-mediated mutagenesis which result in amino acidsubstitutions. Preferably, the variants (including derivatives) encodeless than 50 amino acid substitutions, less than 40 amino acidsubstitutions, less than 30 amino acid substitutions, less than 25 aminoacid substitutions, less than 20 amino acid substitutions, less than 15amino acid substitutions, less than 10 amino acid substitutions, lessthan 5 amino acid substitutions, less than 4 amino acid substitutions,less than 3 amino acid substitutions, or less than 2 amino acidsubstitutions relative to the reference V_(H) region, V_(H)CDR1,V_(H)CDR2, V_(H)CDR3, V_(L) region, V_(L)CDR1, V_(L)CDR2, or V_(L)CDR3.A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a side chain witha similar charge. Families of amino acid residues having side chainswith similar charges have been defined in the art. These familiesinclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g., aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Alternatively, mutations can beintroduced randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forbiological activity to identify mutants that retain activity (e.g., theability to bind a LINGO-1 polypeptide).

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations may alter an antibody's ability to bind antigen. The locationof most silent and neutral missense mutations is likely to be in theframework regions, while the location of most non-neutral missensemutations is likely to be in CDR, though this is not an absoluterequirement. One of skill in the art would be able to design and testmutant molecules with desired properties such as no alteration inantigen binding activity or alteration in binding activity (e.g.,improvements in antigen binding activity or change in antibodyspecificity). Following mutagenesis, the encoded protein may routinelybe expressed and the functional and/or biological activity of theencoded protein, (e.g., ability to immunospecifically bind at least oneepitope of a LINGO-1 polypeptide) can be determined using techniquesdescribed herein or by routinely modifying techniques known in the art.

IV. Polynucleotides Encoding LINGO-1 Antibodies

The present invention also provides for nucleic acid molecules encodingLINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention.

In one embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region(VH), where at least one of the CDRs of the heavy chain variable regionor at least two of the CDRs of the heavy chain variable region are atleast 80%, 85%, 90% or 95% identical to reference heavy chain CDR1,CDR2, or CDR3 amino acid sequences of Li62 or Li81 or variants thereofas described in Table 3. Alternatively, the CDR1, CDR2, and CDR3 regionsof the VH are at least 80%, 85%, 90% or 95% identical to reference heavychain CDR1, CDR2, and CDR3 amino acid sequences of Li62 or Li81 orvariants thereof as described in Table 3. Thus, according to thisembodiment a heavy chain variable region of the invention has CDR1,CDR2, or CDR3 polypeptide sequences related to the polypeptide sequencesshown in Table 3:

TABLE 3 LINGO-1 Antibody VH Sequences VH VH VH Antibody VH SEQUENCE CDR1CDR2 CDR3 Li62 EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGHNDRQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFDLSKNTLYLQMNSLRAEDTATYYCAREGHNDWYFDLW ID DSVKG (SEQ IDGRGTLVTVSS (SEQ ID NO: 1) NO: 2) (SEQ ID NO: 4) NO: 3) Li62EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGYYD variantRQAPGKGLEWVSW-GPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFD B06SKNTLYLQMNSLRAEDTATYYCAREGYYDWYFDQW ID DSVKG Q (SEQGRGTLVTVSS (SEQ ID NO: 53) NO: 2) (SEQ ID ID NO: 3) NO: 17) Li62EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGQYD variantRQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFD B12SKNTLYLQMNSLRAEDTATYYCAREGQYDWYFDVW ID DSVKG V (SEQGRGTLVTVSS (SEQ ID NO: 54) NO: 2) (SEQ ID ID NO: 3) NO: 18) Li62EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGDYD variantRQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFDL F06SKNTLYLQMNSLRAEDTATYYCAREGDYDWYFDLW ID DSVKG (SEQ IDGRGTLVTVSS (SEQ ID NO: 55) NO: 2) (SEQ ID NO: 19) NO: 3) Li62EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGQYD variantRQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFEL B01SKNTLYLQMNSLRAEDTATYYCAREGQYDWYFELW ID DSVKG (SEQ IDGRGTLVTVSS (SEQ ID NO: 56) NO: 2) (SEQ ID NO: 20) NO: 3) Li62EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EADID variantRQAPGKGLEWVSW-GPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WFFDL D09SKNTLYLQMNSLRAEDTATYYCAREADIDWFFDLWG ID DSVKG (SEQ IDRGTLVTVSS (SEQ ID NO: 57) NO: 2) (SEQ ID NO: 21) NO: 3) Li62EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMIWV IYPMF WIGPSG EGHYD variantRQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFDL D12SKNTLYLQMNSLRAEDTATYYCAREGHYDWYFDLW ID DSVKG (SEQ IDGRGTLVTVSS (SEQ ID NO: 58) NO: 2) (SEQ ID NO: 22) NO: 3) Li62EVQLLESGGGLVQPGGSLRLSCAASGETESTYPMEWV IYPMF WIGPSG EGRYD variantRQAPGKGLEWVSWIGPSGGITKYADSVKGREFISRDN (SEQ GITKYA WYFDP F01SKNTIXLQMNSLRAEDTATYYCAREGRYDWYFDPW ID DSVKG (SEQ IDGRGTLVTVSS (SEQ ID NO: 59) NO: 2) (SEQ ID NO: 23) NO: 3) Li62EVQLLESGGGLVQPGGSERLSCAASGFTESEYTMFWV IYPMF WFdPSG EGDYD variantRQAPGKGLEWVSWIGPSGGEFKYADSVKGRFTISIRDN (SEQ GITKYA WYFGL F02SKINTLYLQMNSLRAEDTATYYCAREGDYDWYEGLW ID DSVKG (SEQ IDGRGTLVTVSS (SEQ ID NO: 60) NO: 2) (SEQ ID NO: 24) NO: 3) Li62EVQLLESGGGLVQPGGSLALSCAASGEFFSIYPMFWV IYPMF WIGPSG EGRYD variantRQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFDL F06SKNTLYEQIVINSLRAEDTATYYCAREGRYDWYEDLW ID DSVKG (SEQ IDGRGTLVTVSS (SFQ ID NO: 61) NO: 2) (SEQ ID NO: 25) NO: 3) Li62EVQLLESGGGLVQPGGSERESCAASGFTESIYPMFWV IYPMF WIGPSG ESHID variantRQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA RYFDL F10SKNTLYLQMNSLRAEDTATTYCARESHIDRYFDLWG ID DSVKG (SEQ IDRGTLVTVSS (SEQ ID NO: 62) NO: 2) (SEQ ID NO: 26) NO: 3) Li62EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGQYD variantRQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFD G08SKNTLYLQMNSLRAEDTATYYCAREGQYDWYEDVW ID DSVKG V (SEQGRGTLVIVSS (SEQ ID NO: 63) NO: 2) (SEQ ID ID NO: 3) NO: 27) Li62EVQLLESGGGINQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGITYN variantRQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA GYFDL H08SKNTLATQMNSLRAEDTATYYCAREGHYNGYEDLW ID DSVKG (SEQ IDGRGTLVTVSS (SEQ ID NO: 64) NO: 2) (SEQ ID NO: 28) NO: 3) Li62EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGDYD variantRQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYEDL C10SKNTINLQMNSIAAEDIATYYCAREGYYDWYEDLW ID DSVKG (SEQ IDGRGTLVTVSS (SEQ ID NO: 65) NO: 2) (SEQ ID NO: 29) NO: 3) Li62EVQLLESGGGLVQPGCTSLRLSCAASGETFSTYPMEWV WPMF WIGPSG EGTYD variantRQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYLD C02SKNTLYLQMNSLRAEDTATYYCAREGTYDWYILDLW ID DSVKG L (SEQGRGTLVTVSS (SEQ ID NO: 66) NO: 2) (SEQ ID ID NO: 3) NO: 30) Li62EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGYYD variantRQAPGKGLEWVSWIGPSGGEFKYADSVKGRFTISRDN (SEQ GITKYA WYFEL D05SKNTLYLQMNSLRAEDTATYYCAREGYYDWYFELW ID DSVKG (SEQ IDGRGTLVTVSS (SEQ ID NO: 67) NO: 2) SEQ ID NO: 31) NO: 3) Li62EVQLLESGGGLVQPGGSLRLSCAASGFTESTYPMFWV IYPMF WIGPSG EGLID variantRQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WEFDQ F02SKNTLYLQMNSLRAEDTATYYCAREGLIDWEEDQWG ID DSVKG (SEQ IDRGTLVTVSS (SEQ ID NO: 68) NO: 2) (SEQ ID NO: 32) NO: 3) Li62EVQLLESGGGLVQPGGSLRLSCAASGETESIYPMFWVI IYPMF WIGPSG EGQFD variantRQAPGKGIEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFDL C10SKNTLYLQMNSLRAEDTATYYCAREGQEDWYFDLW ID DSVKG (SEQ IDGRGTLVTVSS (SEQ ID NO: 69) NO: 2) (SEQ ID NO: 33) NO: 3) Li62EVQLLESGGGLVQPGGSLRLSCAASCIFTSIYPMFWV IYPMF WIGPSG EGTYD variantRQAPGKGIEWAISWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYEDL H08SKNTIXLQMNSLRAEDTATYYCAREGTYDWYFDLW ID DSVKG (SEQ IDGRGTLVTVSS (SEQ ID NO: 70) NO: 2) (SEQ ID NO: 34) NO: 3) Li81EVQLLESGGGLVQPGGSLRESCAASGETESAYEMKW AYEM VIGPSG EGDNDVRQAPGKGLEWVSVIGPSGGFTEYADSVKGRFTISRD K (SEQ GFTFY AFDINSKNTLYLQMNSLRAEDTAVYYCATEGDNDAFDIWG ID ADSVK (SEQ IDQGGTVTVSS (SEQ ID NO: 5) NO: 6) G (SEQ NO: 8) ID NO: 7) Li81EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM E VIGPSG EGEND variantVRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AFDV F09NSKNTLYLQMNSLRAEDTAVYYCATEGENDAFDVW ID ADSVK (SEQ IDGQGTTVTVSS (SEQ ID NO:71) NO:6) (SEQ NO: 35) ID NO: 7) Li81EVQLLESGGGLVQPGGSERISCAASGFTFSAYEMKW AYEM VIGPSG EGDND variantVRQAPGKGLEWVSVIGPSGGETEYADSVKGRFTISRD K (SEQ GFTFY AYDT G02NSKNILYLQMNSLRAEDIAVYYCATEGDNDAYDTW ID ADSVK (SEQ IDGQGITVTVSS (SEQ ID NO: 72) NO: 6) G (SEQ NO: 36) ID NO: 7) Li81EVQLLESGGGLVQPGGSLRLSCAASGETFSAYEMKW AYEM VIGPSG FGTND variantVRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AFDI H03NSKNTLYLQMNSLRAEDTAVYYCATEGTNDAFDIWG ID ADSVK (SEQ IDQGTTVTVSS (SEQ ID NO: 73) NO: 6) G (SEQ NO: 37) ID NO: 7) Li81EVQLLESGGGLVQPGGSIRLSCAASGFTFSAYEMKW AYEM VIGPSG EGDND variantVRQAPGKGLEWVSVIGPSGGFTEYADSVKGR K (SEQ GFTFY AFDS A12FTISRDNSKNTLYEQMNSLRAEDTAVYYCATEGDN ID ADSVK (SEQ IDDAFDSWGQGTTVTVSS (SEQ ID NO: 74) NO: 6) G (SEQ NO: 38) ID NO: 7) Li81EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGDND variantVRQAPGKGLEVINSVIGPSGGETFYADSVKGRIETISRD K (SEQ GFTFY AFDT C02NSKNTLYLQMNSLRAEDTAVYYCATEGDNDAFDTW ID ADSVK (SEQ IDGQGTTVTVSS (SEQ ID NO: 75) NO: 6) G (SEQ NO: 39) ID NO: 7) Li81EVQLLFSGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGDND variantVRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AYDR C11NSKNTLYLQMNSLRAEDTAVYYCATEGDNDAYDRW ID ADSVK (SEQ IDGQGTTVTVSS (SEQ ID NO: 76) NO: 6) G (SEQ NO: 40) ID NO: 7) Li81EVQLLESGGGLVQPGGSLRLSCAASGETESAYEMKW AYEM VIGPSG EGDND variantVRQAPGKGLEWVSVIGPSGGFTFYADSVKGRETISRD K (SEQ GFTFY VFDS D11NSKNTLYLQMNSLRAEDTAVYYCATEGDNDVIDSW ID ADSVK (SEQ IDGQGITVIVSS (SEQ ID NO: 77) NO: 6) G (SEQ NO: 41) ID NO: 7) Li81EVQLLESGGGLVQPGGSLRLSCAASGFTESAYEMKW AYEM VIGPSG EGDDD variantVRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY VEDM E05NSKNTLYLQMNSL RAEDTAVYYCATEGDDDVFDMW ID ADSVK (SEQ IDGQG0TVTVSS (SEQ ID NO: 78) NO: 6) G (SEQ NO: 42) ID NO: 7) Li81EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGYND variantVRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AFDF H04NSKNTLYLQMNSLRAEDTAVYYCATEGYNDAFDFW ID ADSVK (SEQ IDGQGITVTVSS (SEQ ID NO: 79) NO:6) G (SEQ NO: 43) ID NO: 7) Li81EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGDDD variantVRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AYDM B04NSKNTLYLQMNSLRAEDTAVYYCATEGDDDAYDMW ID ADSVK (SEQ IDGQGTTVTVSS (SEQ ID NO: 80) NO: 6) G (SEQ NO: 44) ID NO: 7) Li81EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EQDYD variantVRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY TYDL A02NSKNTLYLQMNSLRAEDTAVYYCATEQDYDTYDLW ID ADSVK (SEQ IDGQGTTVTVSS (SEQ ID NO: 81) NO: 6) G (SEQ NO: 45) ID NO: 7) Li81EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGDDD variantVRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AFDT B12NSKNTLYLQMNSLRAEDTAVYYCATEGDDDAFDTW ID ADSVK (SEQ IDGQGTTVTVSS (SEQ ID NO: 82) NO: 6) G (SEQ NO: 46) ID NO: 7) Li81EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EADDD variantVRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AFDI H06NSKNTLYLQMNSLRAEDTAVYYCATEADDDAFDIWG ID ADSVK (SEQ IDQGTTVTVSS (SEQ ID NO: 83) NO: 6) G (SEQ NO: 47) ID NO: 7) Li81EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGEND variantVRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AFDM H08NSKNTLYLQMNSLRAEDTAVYYCATEGENDAFDMW ID ADSVK (SEQ IDGQGTTVTVSS (SEQ ID NO: 84) NO: 6) G (SEQ NO: 48) ID NO: 7) Li81EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGEYD variantVRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY TYDI E07NSKNTLYLQMNSLRAEDTAVYYCATEGEYDTYDIWG ID ADSVK (SEQ IDQGTTVTVSS (SEQ ID NO: 85) NO: 6) G (SEQ NO: 9) ID NO: 7)

In certain embodiments, the present invention provides an isolatedpolynucleotide, comprising, consisting essentially of, or consisting ofa nucleic acid encoding an immunoglobulin heavy chain which is identicalto the polypeptide of SEQ ID NO:146 except for a replacement of one ormore of the following amino acids: W50, P53, I57 and/or W104. In someembodiments, W50 is replaced with an H, F, L, M, G, I, or D residue. Insome embodiments, P53 is replaced with an L, S, T, W, or G residue. Insome embodiments, I57 is replaced with a G, M, N, H, L, F, W, Y, S, P, Vor T residue. In some embodiments, W104 is replaced with a V, H, S, Q,M, L, T, or I residue.

In certain embodiments, the present invention provides an isolatedpolynucleotide, comprising, consisting essentially of, or consisting ofa nucleic acid encoding an immunoglobulin heavy chain variable regionwhich is identical to the polypeptide of SEQ ID NO:5 except for areplacement of amino acid P53. In some embodiments, P53 is replaced withan L, S, T, W, or G residue.

In certain embodiments, the present invention provides an isolatedpolynucleotide, comprising, consisting essentially of, or consisting ofa nucleic acid encoding an immunoglobulin heavy chain varible regionwhich is identical to the polypeptide of SEQ ID NO:1 except for areplacement of one or more of the following amino acids: W50, P53, I57and/or W104. In some embodiments, W50 is replaced with an H, F, L, M, G,I, or D residue. In some embodiments, P53 is replaced with an L, S, T,W, or G residue. In some embodiments, I57 is replaced with a G, M, N, H,L, F, W, Y, S, P, V or T residue. In some embodiments, W104 is replacedwith a V, H, S, Q, M, L, T, or I residue.

In certain embodiments, the present invention provides an isolatedpolynucleotide, comprising, consisting essentially of, or consisting ofa nucleic acid encoding en immunoglobulin heavy chain variable regionwhich is identical to the polypeptide of SEQ ID NO:66 except for areplacement of one or more of the following amino acids: W50, P53, I57and/or W104. In some embodiments, W50 is replaced with an H, F, L, M, G,I, or D residue. In some embodiments, P53 is replaced with an L, 5, T,W, or G residue. In some embodiments, I57 is replaced with a Q, M, N, H,L, W, Y, S, P, V or T residue. In some embodiments, W104 is replacedwith a V, H, S, Q, M, L, T, or I residue.

In certain embodiments, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region (VH)in which the CDR3 region has a polypeptide sequence at least 80%, 85%,90%, 95% or 100% identical to the CDR3 amino acid sequences selectedfrom the group consisting of SEQ ID NOs: 4, 8 and 17-49. In someembodiments, the CDR1 and CDR2 regions are at least 80%, 85%, 90%, 95%or 100% identical to the CDR1 and CDR2 amino acid sequences of SEQ IDNOs: 2 and 3, respectively, and the CDR3 region is at least 80%, 85%,90%, 95% or 100% identical to a CDR3 amino acid sequence selected fromthe group consisting of SEQ ID NOs: 4 and 17-34. In some embodiments,the CDR1 and CDR2 regions are at least 80%, 85%, 90%, 95% or 100%identical to the CDR1 and CDR2 amino acid sequences of SEQ ID NOs: 6 and7, respectively, and the CDR3 region is at least 80%, 85%, 90%, 95% or100% identical to a CDR3 amino acid sequence selected from the groupconsisting of SEQ ID NOs: 8 and 35-49.

In certain embodiments, an antibody or antigen-binding fragmentcomprising the VH encoded by the polynucleotide specifically orpreferentially binds to LINGO-1.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin heavy chain variable region (VH)in which the CDR1, CDR2, and CDR3 regions have polypeptide sequenceswhich are identical to the CDR1, CDR2, and CDR3 groups shown in Table 3.In certain embodiments, an antibody or antigen-binding fragmentcomprising the VH encoded by the polynucleotide specifically orpreferentially binds to LINGO-1.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VH encoded byone or more of the polynucleotides described above specifically orpreferentially binds to the same epitope as a Li62 or Li81 antibody, orwill competitively inhibit such a monoclonal antibody from binding toLINGO-1.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VH encoded byone or more of the polynucleotides described above specifically orpreferentially binds to a LINGO-1 polypeptide or fragment thereof, or aLINGO-1 variant polypeptide, with an affinity characterized by adissociation constant (K_(D)) no greater than 5×10⁻² M, 10⁻² M, 5×10⁻³M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶M, 10⁻⁶ M, 5×10⁻⁷M,10⁻⁷ M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹M,10⁻¹¹M, 5×10⁻¹²M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10¹⁴ M,5×10⁻¹⁵ M, or 10⁻⁵ M.

in a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a VH at least 80%, 85%, 90% or 95% identical to areference VH polypeptide selected from the group consisting of SEQ IDNOs: 1, 5 and 53-85. In certain embodiments, an antibody orantigen-binding fragment comprising the VH encoded by the polynucleotidespecifically or preferentially binds to LINGO-1

In some embodiments, the isolated polynucleotide comprises, consistsessentially of or consists of a nucleic acid encoding an antibody heavychain as shown below in SEQ ID NO:86.

(SEQ ID NO: 86)EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKWVRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYVATEGDNDAFDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG

In other embodiments, the isolated polynucleotide comprises, consistsessentially of or consists of a nucleic acid encoding an aglycosylatedversion of an antibody heavy chain. For example, an aglycosylatedversion of Li81 is described in PCT/US2008/000316, fled Jan. 9, 2008,which is incorporated herein by reference in its entirety. Anaglycosylated version of the Li8I antibody was created by changing asingle amino acid (T to A) in the Li81 heavy chain sequence. Thesequence of an aglycosylated version of Li81 heavy chain (SEQ ID NO:50)is shown below. The single amino acid change is marked in bold andunderlined:

(SEQ ID NO: 50)EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKWVRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATEGDNDAFDPWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGCITAALGCLVKDYFPEPVTVSWNSGALTSGVHTEPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKINWYVDCiVEVHNAKTKPREEQYNS A YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELIKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTFPPVLDSDGSFINSKLTVDKSRWQQGNVTSCSVMHEALHNHYT QKSLSLSPG.

Therefore, the present invention includes an isolated polynucleotidecomprising, consisting essentially of, or consisting of a nucleic acidencoding a heavy chain at least 80%, 85%, 90% or 95% identical to areference polypeptide comprising the amino acids of SEQ ID NO:50 or 86.In certain embodiments, an antibody or antigen-binding fragmentcomprising the heavy chain encoded by the polynucleotide specifically orpreferentially binds to LINGO-1.

In another aspect, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of or consisting of anucleic acid sequence encoding a VH comprising the amino acids of SEQ IDNO: 1 or SEQ ID NO: 5. In certain embodiments, an antibody orantigen-binding fragment comprising the VH encoded by the polynucleotidespecifically or preferentially binds to LINGO-1. In certain embodiments,an antibody or antigen-binding fragment thereof comprising, consistingessentially of or consisting of a VH encoded by one or more of thepolynucleotides described above specifically or preferentially binds tothe same epitope as Li62, Li81 or a variant thereof as described inTable 3 or will competitively inhibit such a monoclonal antibody frombinding to LINGO-1.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VH encoded byone or more of the polynucleotides described above specifically orpreferentially binds to a LINGO-1 polypeptide or fragment thereof, or aLINGO-1 variant polypeptide, with an affinity characterized by adissociation constant (K_(D)) no greater than 5×10⁻² M, 10⁻² M, 5×10⁻³M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, M,5×M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M,5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹² M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M,or 10⁻¹⁵ M.

VL Sequences

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding an immunoglobulin light chain variable region(VL), where at least one of the CDRs of the light chain variable regionor at least two of the CDRs of the light chain variable region are atleast 80%, 85%, 90% or 95% identical to reference light chain CDR1,CDR2, or CDR3 amino acid sequences from monoclonal LINGO-1 antibodiesdisclosed herein. Alternatively, the CDR1, CDR2, and CDR3 regions of theVL are at least 80%, 85%, 90% or 95% identical to reference light chainCDR1, CDR2, and CDR3 amino acid sequences from monoclonal LINGO-1antibodies disclosed herein. Thus, according to one embodiment, a lightchain variable region of the invention has CDR1, CDR2, or CDR3polypeptide sequences related to the polypeptide sequences shown inTable 4.

TABLE 4 LINGO-1 Antibody VL Sequences VL VL VL Antibody VL SEQUENCE CDR1CDR2 CDR3 Li62 DIQMTQSPSFLSASVGDSVAITCRASQDISRYLAWYQQ RASQD DASNL QQYDTRPGKAPKLLIYDASNLQTGVPSRFSGSGSGTDFTFTITS ISRYL QT (SEQ LHPSLQPEDFGTYYCQQYDTLHPSFGPGTTVDIK (SEQ ID A (SEQ ID (SEQ ID NO: 9) IDNO: 11) NO: 12) NO: 10) Li81 DIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ RASQSDASNR QQRSN QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS VSSYL AT (SEQ WPMYSLEPEDFAVYYCQQRSNWPMYTFGQGTKLEIK (SEQ A (SEQ ID NO: T (SEQ ID NO:13) ID15) ID NO: NO: 14) 16)

In certain embodiments, an antibody or antigen-binding fragmentcomprising the VL encoded by the polynucleotide specifically orpreferentially binds to LINGO-1.

In certain embodiments, the present invention provides an isolatedpolynucleotide, comprising, consisting essentially of or consisting of anucleic acid encoding an immunoglobulin light chain which is identicalto the polypeptide of SEQ ID NO:145 except for a replacement of aminoacid W94. In some embodiments, W94 is replaced with an A, D, L, N, G, Q,V, or S residue.

In certain embodiments, the present invention provides an isolatedpolynucleotide, comprising, consisting essentially of, or consisting ofa nucleic acid encoding an immunoglobulin light chain variable regionwhich is identical to the polypeptide of SEQ ID NO:5 except for areplacement of amino acid W94. In some embodiments, W94 is replaced withan A, D, L, N, G, Q, V, or S residue.

In another embodiment, the present invention provides an isolatedpolynucleotide comprising, consisting essentially of or consisting of anucleic acid encoding an immunoglobulin light chain variable region (VL)in which the CDR1, CDR2, and CDR3 regions have polypeptide sequenceswhich are identical to the CDR1, CDR2, and CDR3 groups shown in Table 4.In certain embodiments, an antibody or antigen-binding fragmentcomprising the VL encoded by the polynucleotide specifically orpreferentially binds to LINGO-1.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VL encoded byone or more of the polynucleotides described above specifically orpreferentially binds to the same epitope as Li62 or Li81, or willcompetitively inhibit such an antibody from binding to LINGO-1.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VL encoded byone or more of the polynucleotides described above specifically orpreferentially binds to a LINGO-1 polypeptide or fragment thereof, or aLINGO-1 variant polypeptide, with an affinity characterized by adissociation constant (K_(D)) no greater than 5×10⁻² M, 10⁻² M, 5×10⁻³M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷M, 10⁻⁷ M, 5×10⁻⁷ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M,5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M,10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

In a further embodiment, the present invention includes an isolatedpolynucleotide comprising, consisting essentially of, or consisting of anucleic acid encoding a VL at least 80%, 85%, 90% or 95% identical to areference VL polypeptide sequence selected from SEQ ID NO: 9 or SEQ IDNO: 13. In certain embodiments, an antibody or antigen-binding fragmentcomprising the VL encoded by the polynucleotide specifically orpreferentially binds to LINGO-1. In another aspect, the presentinvention includes an isolated polynucleotide comprising, consistingessentially of, or consisting of a nucleic acid sequence encoding a VLof the invention, selected from SEQ ID NO: 9 or SEQ ID NO: 13. Incertain embodiments, an antibody or antigen-binding fragment comprisingthe VL encoded by the polynucleotide specifically or preferentiallybinds to LINGO-1. In certain embodiments, an antibody or antigen-bindingfragment thereof comprising, consisting essentially of, or consisting ofa VL encoded by one or more of the polynucleotides described abovespecifically or preferentially binds to the same epitope as Li62 orLi81, or will competitively inhibit such a monoclonal antibody frombinding to LINGO-1.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a VL encoded byone or more of the polynucleotides described above specifically orpreferentially binds to a LINGO-1 polypeptide or fragment thereof, or aLINGO-1 variant polypeptide, with an affinity characterized by adissociation constant (K_(D)) no greater than 5×10⁻² M, 10⁻² M, 5×10⁻³M, 10⁻³ M, 5×10⁻⁴ M, 10⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M,5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M,10⁴⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

Any of the polynucleotides described above may further includeadditional nucleic acids, encoding, e.g., a signal peptide to directsecretion of the encoded polypeptide, antibody constant regions asdescribed herein, or other heterologous polypeptides as describedherein.

Also, as described in more detail elsewhere herein, the presentinvention includes compositions comprising the polynucleotidescomprising one or more of the polynucleotides described above. In oneembodiment, the invention includes compositions comprising a firstpolynucleotide and second polynucleotide wherein said firstpolynucleotide encodes a VH polypeptide as described herein and whereinsaid second polynucleotide encodes a VL polypeptide as described herein.

The present invention also includes fragments of the polynucleotides ofthe invention, as described elsewhere. Additionally polynucleotideswhich encode fusion polynucleotides, Fab fragments, and otherderivatives, as described herein, are also contemplated by theinvention.

The polynucleotides may be produced or manufactured by any method knownin the art. For example, if the nucleotide sequence of the antibody isknown, a polynucleotide encoding the antibody may be assembled fromchemically synthesized oligonucleotides (e.g., as described in Kutmeieret al., BioTechniques 17:242 (1994)), which, briefly, involves thesynthesis of overlapping oligonucleotides containing portions of thesequence encoding the antibody, annealing and ligating of thoseoligonucleotides, and then amplification of the ligated oligonucleotidesby PCR.

Alternatively, a polynucleotide encoding a LINGO-1 antibody, orantigen-binding fragment, variant, or derivative thereof may begenerated from nucleic acid from a suitable source. If a clonecontaining a nucleic acid encoding a particular antibody is notavailable, but the sequence of the antibody molecule is known, a nucleicacid encoding the antibody may be chemically synthesized or obtainedfrom a suitable source (e.g., an antibody cDNA library, or a cDNAlibrary generated from, or nucleic acid, preferably poly A+RNA, isolatedfrom, any tissue or cells expressing the antibody or other LINGO-1antibody, such as hybridoma cells selected to express an antibody) byPCR amplification using synthetic primers hybridizable to the 3′ and 5′ends of the sequence or by cloning using an oligonucleotide probespecific for the particular gene sequence to identify, e.g., a cDNAclone from a cDNA library that encodes the antibody or other LINGO-1antibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe LINGO-1 antibody, or antigen-binding fragment, variant, orderivative thereof is determined, its nucleotide sequence may bemanipulated using methods well known in the art for the manipulation ofnucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y. (1990) and Ausubel etal., eds., Current Protocols in Molecular Biology, John Wiley & Sons, NY(1998), which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

A polynucleotide encoding a LINGO-1 antibody, or antigen-bindingfragment, variant, or derivative thereof can be composed of anypolyribonucleotide or polydeoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, a polynucleotideencoding LINGO-1 antibody, or antigen-binding fragment, variant, orderivative thereof can be composed of single- and double-stranded DNA,DNA that is a mixture of single- and double-stranded regions, single-and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. In addition, a polynucleotideencoding a LINGO-1 antibody, or antigen-binding fragment, variant, orderivative thereof can be composed of triple-stranded regions comprisingRNA or DNA or both RNA and DNA. A polynucleotide encoding a LINGO-1antibody, or antigen-binding fragment, variant, or derivative thereofmay also contain one or more modified bases or DNA or RNA backbonesmodified for stability or for other reasons. “Modified” bases include,for example, tritylated bases and unusual bases such as inosine. Avariety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

An isolated polynucleotide encoding a non-natural variant of apolypeptide derived from an immunoglobulin (e.g., an immunoglobulinheavy chain portion or light chain portion) can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of the immunoglobulin such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations may be introduced by standard techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conservative amino acid substitutions are made at one ormore non-essential amino acid residues.

V. LINGO-1 Antibody Polypeptides

The present invention is further directed to isolated polypeptides whichmake up LINGO-1 antibodies, antigen binding fragments, variants orderivatives thereof. LINGO-1 antibodies of the present inventioncomprise polypeptides, e.g., amino acid sequences encodingLINGO-1-specific antigen binding regions derived from immunoglobulinmolecules. A polypeptide or amino acid sequence “derived from” adesignated protein refers to the origin of the polypeptide. In certaincases, the polypeptide or amino acid sequence which is derived from aparticular starting polypeptide or amino acid sequence has an amino acidsequence that is essentially identical to that of the starting sequence,or a portion thereof, wherein the portion consists of at least 10-20amino acids, at least 20-30 amino acids, at least 30-50 amino acids, orwhich is otherwise identifiable to one of ordinary skill in the art ashaving its origin in the starting sequence.

In one embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (VH), where at least one ofCDRs of the heavy chain variable region or at least two of the CDRs ofthe heavy chain variable region are at least 80%, 85%, 90% or 95%identical to reference heavy chain CDR1, CDR2 or CDR3 amino acidsequences from monoclonal LINGO-1 antibodies disclosed herein.Alternatively, the CDR1, CDR2 and CDR3 regions of the VH are at least80%, 85%, 90% or 95% identical to reference heavy chain CDR1, CDR2 andCDR3 amino acid sequences from monoclonal LINGO-1 antibodies disclosedherein. Thus, according to this embodiment a heavy chain variable regionof the invention has CDR1, CDR2, and CDR3 polypeptide sequences relatedto the groups shown in Table 3, supra. In certain embodiments, anantibody or antigen-binding fragment comprising the VH polypeptidespecifically or preferentially binds to LINGO-1.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (VH), wherein at least theCDR3 region is at least 80%, 85%, 90% or 95% identical to a referenceCDR3 sequence selected from the group consisting of SEQ ID NOs: 4, 8 and17-49. In further embodiments, the CDR3 region is identical to areference CDR3 sequence selected from the group consisting of SEQ IDNOs: 4, 8 and 17-49. In still further embodiments, the inventionprovides an isolated polypeptide comprising, consisting essentially of,or consisting of an immunoglobulin heavy chain variable region (VH),wherein, the CDR1 and CDR2 regions are at least 80%, 85%, 90%, 95% or100% identical to the CDR1 and CDR2 amino acid sequences of SEQ ID NOs:2 and 3, respectively, and the CDR3 region is at least 80%, 85%, 90%,95% or 100% identical to a CDR3 amino acid sequence selected from thegroup consisting of SEQ ID NOs: 4 and 17-34. In other embodiments, theinvention provides an isolated polypeptide comprising, consistingessentially of, or consisting of an immunoglobulin heavy chain variableregion (VH), wherein the CDR1 and CDR2 regions are at least 80%, 85%,90%, 95% or 100% identical to the CDR1 and CDR2 amino acid sequences ofSEQ ID NOs: 6 and 7, respectively, and the CDR3 region is at least 80%,85%, 90%, 95% or 100% identical to a CDR3 amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 8 and 35-49.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region (VH) in which the CDR1, CDR2,and CDR3 regions have polypeptide sequences which are identical to theCDR1, CDR2, and CDR3 groups shown in Table 3. In certain embodiments, anantibody or antigen-binding fragment comprising the VH polypeptidespecifically or preferentially binds to LINGO-1.

In a further embodiment, the present invention includes an isolatedpolypeptide comprising, consisting essentially of, or consisting of a VHpolypeptide at least 80%, 85%, 90% 95% or 100% identical to a referenceVH polypeptide sequence selected from SEQ ID NOs: 1, 5 and 53-85. In oneparticular embodiment, the VH polypeptide comprises a CDR3 amino acidsequence selected from the group consisting of SEQ ID NOs: 4, 8 and17-49.

In certain embodiments, an antibody or antigen-binding fragmentcomprising the VH polypeptide specifically or preferentially binds toLINGO-1. In another aspect, the present invention includes an isolatedpolypeptide comprising, consisting essentially of, or consisting of a VHpolypeptide selected from the group consisting of SEQ ID NOs: 1, 5 and53-85. In certain embodiments, an antibody or antigen-binding fragmentcomprising the VH polypeptide specifically or preferentially binds toLINGO-1.

In certain embodiments, the present invention provides an isolatedpolypeptide, comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain which is identical to the polypeptide of SEQID NO:146 except for a replacement of one or more of the following aminoacids: W50, P53, I57 and/or W104. In some embodiments, W50 is replacedwith an H, F, L, M, G, I, or D residue. In some embodiments, P53 isreplaced with an L, S, T, W, or G residue. In some embodiments, I57 isreplaced with a G, M, N, H, L, F, W, Y, S, P, V or T residue. In someembodiments, W104 is replaced with a V, H, S, Q, M, L, T, or I residue.

In certain embodiments, the present invention provides an isolatedpolypeptide, comp sing, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region which is identical to thepolypeptide of SEQ ID NO:5 except for a replacement of amino acid P53.In some embodiments, P53 is replaced with an L, S, T, W, or G residue.

In certain embodiments, the present invention provides an isolatedpolypeptide, comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain varible region which is identical to thepolypeptide of SEQ ID NO:1 except for a replacement of one or more ofthe following amino acids: W50, P53, I57 and/or W104. In someembodiments, W50 is replaced with an H, F, L, M, G, I, or D residue. Insome embodiments, P53 is replaced with an L, S, T, W, or G residue. Insome embodiments, I57 is replaced with a G, M, N, H, L, F, W, Y, S, P, Vor T residue. In some embodiments, W104 is replaced with a V, H, S, Q,M, L, T, or I residue.

In certain embodiments, the present invention provides an isolatedpolypeptide, comprising, consisting essentially of, or consisting of animmunoglobulin heavy chain variable region which is identical to thepolypeptide of SEQ ID NO:66 except for a replacement of one or more ofthe following amino acids: W50, P53, I57 and/or W104. In someembodiments, W50 is replaced with an H, F, L, M, G, I, or D residue. Insome embodiments, P53 is replaced with an L, S, T, W, or G residue. Insome embodiments, I57 is replaced with a G, M, N, H, L, F, W, Y, S, P, Vor T residue. In some embodiments, W104 is replaced with a V, H, S, Q,M, L, T, or I residue.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a one or more ofthe VH polypeptides described above specifically or preferentially bindsto the same epitope as Li62, Li81 or a variant thereof as described inTable 3, or will competitively inhibit such an antibody from binding toLINGO-1.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of one or more ofthe VH polypeptides described above specifically or preferentially bindsto a LINGO-1 polypeptide or fragment thereof, or a LINGO-1 variantpolypeptide, with an affinity characterized by a dissociation constant(K_(D)) no greater than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M,10⁻⁴ M, 5×10⁻⁵M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, M, 5×10⁻⁸ M, 10⁻⁴M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10¹² M,10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (VL), where at least one ofthe CDRs of the light chain variable region or at least two of the CDRsof the light chain variable region are at least 80%, 85%, 90% or 95%identical to reference heavy chain CDR1, CDR2, or CDR3 amino acidsequences from monoclonal LINGO-1 antibodies disclosed herein.Alternatively, the CDR1, CDR2 and CDR3 regions of the VL are at least80%, 85%, 90% or 95% identical to reference light chain CDR1, CDR2, andCDR3 amino acid sequences from monoclonal LINGO-1 antibodies disclosedherein. Thus, according to this embodiment a light chain variable regionof the invention has CDR1, CDR2, and CDR3 polypeptide sequences relatedto the polypeptides shown in Table 4, supra. In certain embodiments, anantibody or antigen-binding fragment comprising the VL polypeptidespecifically or preferentially binds to LINGO-1.

In another embodiment, the present invention provides an isolatedpolypeptide comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region (VL) in which the CDR1, CDR2,and CDR3 regions have polypeptide sequences which are identical to theCDR1, CDR2, and CDR3 groups shown in Table 4. In certain embodiments, anantibody or antigen-binding fragment comprising the VL polypeptidespecifically or preferentially binds to LINGO-1.

In a further embodiment, the present invention includes an isolatedpolypeptide comprising, consisting essentially of, or consisting of a VLpolypeptide at least 80%, 85%, 90% or 95% identical to a reference VLpolypeptide sequence selected from SEQ ID NO: 9 or SEQ ID NO: 13, shownin Table 4. In certain embodiments, an antibody or antigen-bindingfragment comprising the VL polypeptide specifically or preferentiallybinds to LINGO-1. In another aspect, the present invention includes anisolated polypeptide comprising, consisting essentially of, orconsisting of a VL polypeptide selected from SEQ ID NO: 9 or SEQ ID NO:13, shown in Table 4. In certain embodiments, an antibody orantigen-binding fragment comprising the VL polypeptide specifically orpreferentially binds to LINGO-1.

In certain embodiments, the present invention provides an isolatedpolypeptide consisting essentially of, or consisting of animmunoglobulin light chain which is identical to the polypeptide of SEQID NO:145 except for a replacement of amino acid W94. In someembodiments, W94 is replaced with an A, D, L, N, G, Q, V, or S residue.

In certain embodiments, the present invention provides an isolatedpolypeptide, comprising, consisting essentially of, or consisting of animmunoglobulin light chain variable region which is identical to thepolypeptide of SEQ ID NO:5 except for a replacement of amino acid W94.In some embodiments, W94 is replaced with an A, D, L, N, G, Q, V, or Sresidue.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, one or more of the VLpolypeptides described above specifically or preferentially binds to thesame epitope as Li62 or Li81, or will competitively inhibit such amonoclonal antibody from binding to LINGO-1.

In certain embodiments, an antibody or antigen-binding fragment thereofcomprising, consisting essentially of, or consisting of a one or more ofthe VL polypeptides described above specifically or preferentially bindsto a LINGO-1 polypeptide or fragment thereof, or a LINGO-1 variantpolypeptide, with an affinity characterized by a dissociation constant(K_(D)) no greater than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M,10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M, 5×10 M, 10⁻⁷ M, 5×10⁻⁸M,10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹ M, 5×10⁻¹²M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵M.

In other embodiments, an antibody or antigen-binding fragment thereofcomprises, consists essentially of or consists of a VH polypeptide, asshown in Table 3, and a VL polypeptide, as shown in Table 4 selectedfrom the group consisting of:

-   -   i) SEQ ID NO: 1 or SEQ ID NOs: 53-70 and SEQ ID NO: 9; and    -   iii) SEQ ID NO: 5 or SEQ ID NOs: 71-85 and SEQ ID NO: 13.

Any of the polypeptides described above may further include additionalpolypeptides, e.g., a signal peptide to direct secretion of the encodedpolypeptide, antibody constant regions as described herein, or otherheterologous polypeptides as described herein. Additionally,polypeptides of the invention include polypeptide fragments as describedelsewhere. Additionally polypeptides of the invention include fusionpolypeptide, Fab fragments, and other derivatives, as described herein.

Also, as described in more detail elsewhere herein, the presentinvention includes compositions comprising the polypeptides describedabove.

It will also be understood by one of ordinary skill in the art thatLINGO-1 antibody polypeptides as disclosed herein may be modified suchthat they vary in amino acid sequence from the naturally occurringbinding polypeptide from which they were derived. For example, apolypeptide or amino acid sequence derived from a designated protein maybe similar, e.g., have a certain percent identity to the startingsequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, or 95% identicalto the starting sequence.

Furthermore, nucleotide or amino acid substitutions, deletions, orinsertions leading to conservative substitutions or changes at“non-essential” amino acid regions may be made. For example, apolypeptide or amino acid sequence derived from a designated protein maybe identical to the starting sequence except for one or more individualamino acid substitutions, insertions, or deletions, e.g., one, two,three, four, five, six, seven, eight, nine, ten, fifteen, twenty or moreindividual amino acid substitutions, insertions, or deletions. Incertain embodiments, a polypeptide or amino acid sequence derived from adesignated protein has one to five, one to ten, one to fifteen, or oneto twenty individual amino acid substitutions, insertions, or deletionsrelative to the starting sequence.

Certain LINGO-1 antibody polypeptides of the present invention comprise,consist essentially of, or consist of an amino acid sequence derivedfrom a human amino acid sequence. However, certain LINGO-1 antibodypolypeptides comprise one or more contiguous amino acids derived fromanother mammalian species. For example, a LINGO-1 antibody of thepresent invention may include a primate heavy chain portion, hingeportion, or antigen binding region. In another example, one or moremurine-derived amino acids may be present in a non-murine antibodypolypeptide, e.g., in an antigen binding site of a LINGO-1 antibody. Incertain therapeutic applications, LINGO-1-specific antibodies, orantigen-binding fragments, variants, or analogs thereof are designed soas to not be immunogenic in the animal to which, the antibody isadministered.

In certain embodiments, a LINGO-1 antibody polypeptide comprises anamino acid sequence or one or more moieties not normally associated withan antibody. Exemplary modifications are described in more detail below.For example, a single-chain fv antibody fragment of the invention maycomprise a flexible linker sequence, or may be modified to add afunctional moiety (e.g., PEG, a drug, a toxin, or a label).

A LINGO-1 antibody polypeptide of the invention may comprise, consistessentially of, or consist of a fusion protein. Fusion proteins arechimeric molecules which comprise, for example, an immunoglobulinantigen-binding domain with at least one target binding site, and atleast one heterologous portion, i.e., a portion with which it is notnaturally linked in nature. The amino acid sequences may normally existin separate proteins that are brought together in the fusion polypeptideor they may normally exist in the same protein but are placed in a newarrangement in the fusion polypeptide. Fusion proteins may be created,for example, by chemical synthesis, or by creating and translating apolynucleotide in which the peptide regions are encoded in the desiredrelationship.

The term “heterologous” as applied to a polynucleotide or a polypeptide,means that the polynucleotide or polypeptide is derived from a distinctentity from that of the rest of the entity to which it is beingcompared. For instance, as used herein, a “heterologous polypeptide” tobe fused to a LINGO-1 antibody, or an antigen-binding fragment, variant,or analog thereof is derived from a non-immunoglobulin polypeptide ofthe same species, or an immunoglobulin or non-immunoglobulin polypeptideof a different species.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a nonessential amino acidresidue in an immunoglobulin polypeptide is preferably replaced withanother amino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members.

Alternatively, in another embodiment, mutations may be introducedrandomly along all or part of the immunoglobulin coding sequence, suchas by saturation mutagenesis, and the resultant mutants can beincorporated into LINGO-1 antibodies for use in the diagnostic andtreatment methods disclosed herein and screened for their ability tobind to the desired antigen, e.g., LINGO-1.

VI. Fusion Proteins and Antibody Conjugates

As discussed in more detail elsewhere herein, LINGO-1 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention may further be recombinantly fused to a heterologouspolypeptide at the N- or C-terminus or chemically conjugated (includingcovalent and non-covalent conjugations) to polypeptides or othercompositions. For example, LINGO-1-specific LINGO-1 antibodies may berecombinantly fused or conjugated to molecules useful as labels indetection assays and effector molecules such as heterologouspolypeptides, drugs, radionuclides, or toxins. See, e.g., PCTpublications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387, which are incorporated herein by reference intheir entireties.

LINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention include derivatives that aremodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodybinding LINGO-1. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc. Any of numerous chemical modifications may be carried out by knowntechniques, including, but not limited to specific chemical cleavage,acetylation, formylation, metabolic synthesis of tunicamycin, etc.Additionally, the derivative may contain one or more non-classical aminoacids.

LINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. LINGO-1-specific antibodies may be modified bynatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in the LINGO-1-specific antibody,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini, or on moieties such as carbohydrates. It will beappreciated that the same type of modification may be present in thesame or varying degrees at several sites in a given LINGO-1-specificantibody. Also, a given LINGO-1-specific antibody may contain many typesof modifications. LINGO-1-specific antibodies may be branched, forexample, as a result of ubiquitination, and they may be cyclic, with orwithout branching. Cyclic, branched, and branched cyclicLINGO-1-specific antibodies may result from posttranslation naturalprocesses or may be made by synthetic methods. Modifications includeacetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination. (See, for instance, Proteins—StructureAnd Molecular Properties, T. E. Creighton, W.H. Freeman and Company, NewYork 2nd Ed., (1993); Posttranslational Covalent Modification OfProteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 1-12(1983); Seifter et al., Meth Enzymol 182:626-646 (1990); Rattan et al.,Ann NY Acad Sci 663:48-62 (1992)).

The present invention also provides for fusion proteins comprising aLINGO-1 antibody, or antigen-binding fragment, variant, or derivativethereof, and a heterologous polypeptide. The heterologous polypeptide towhich the antibody is fused may be useful for function or is useful totarget the LINGO-1 polypeptide expressing cells. In one embodiment, afusion protein of the invention comprises, consists essentially of, orconsists of, a polypeptide having the amino acid sequence of any one ormore of the V_(H) regions of an antibody of the invention or the aminoacid sequence of any one or more of the V_(L) regions of an antibody ofthe invention or fragments or variants thereof, and a heterologouspolypeptide sequence. In another embodiment, a fusion protein for use inthe diagnostic and treatment methods disclosed herein comprises,consists essentially of, or consists of a polypeptide having the aminoacid sequence of any one, two, three of the V_(H) CDRs of aLINGO-1-specific antibody, or fragments, variants, or derivativesthereof, or the amino acid sequence of any one, two, three of the V_(L)CDRs of a LINGO-1-specific antibody, or fragments, variants, orderivatives thereof, and a heterologous polypeptide sequence. In oneembodiment, the fusion protein comprises a polypeptide having the aminoacid sequence of a V_(H) CDR3 of a LINGO-1-specific antibody of thepresent invention, or fragment, derivative, or variant thereof, and aheterologous polypeptide sequence, which fusion protein specificallybinds to at least one epitope of LINGO-1. In another embodiment, afusion protein comprises a polypeptide having the amino acid sequence ofat least one V_(H) region of a LINGO-1-specific antibody of theinvention and the amino acid sequence of at least one V_(L) region of aLINGO-1-specific antibody of the invention or fragments, derivatives orvariants thereof, and a heterologous polypeptide sequence. Preferably,the V_(H) and V_(L) regions of the fusion protein correspond to a singlesource antibody (or scFv or Fab fragment) which specifically binds atleast one epitope of LINGO-1. In yet another embodiment, a fusionprotein for use in the diagnostic and treatment methods disclosed hereincomprises a polypeptide having the amino acid sequence of any one, two,three or more of the V_(H) CDRs of a LINGO-1-specific antibody and theamino acid sequence of any one, two, three or more of the V_(L) CDRs ofa LINGO-1-specific antibody, or fragments or variants thereof, and aheterologous polypeptide sequence. Preferably, two, three, four, five,six, or more of the V_(L)CDR(s) or V_(L)CDR(s) correspond to singlesource antibody (or scFv or Fab fragment) of the invention. Nucleic acidmolecules encoding these fusion proteins are also encompassed by theinvention.

Exemplary fusion proteins reported in the literature include fusions ofthe T cell receptor (Gascoigne et al., Proc. Natl. Acad. Sci. USA84:2936-2940 (1987)); CD4 (Capon et al., Nature 337:525-531 (1989);Traunecker et al., Nature 339:68-70 (1989); Zettmeissl et al., DNA CellBiol. USA 9:347-353 (1990); and Byrn et al., Nature 344:667-670 (1990));L-selectin (homing receptor) (Watson et al., J. Cell. Biol.110:2221-2229 (1990); and Watson et al., Nature 349:164-167 (1991));CD44 (Aruffo et al., Cell 61:1303-1313 (1990)); CD28 and B7 (Linsley etal., J. Exp. Med. 173:721-730 (1991)); CTLA-4 (Lisley et al., Exp. Med.174:561-569 (1991)); CD22 (Stamenkovic et al., Cell 66:1133-1144(1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol. 27:2883-2886(1991); and Peppel et al., J. Exp. Med. 174:1483-1489 (1991)); and IgEreceptor a (Ridgway and Gorman, J. Cell. Biol. Vol. 115, Abstract No.1448 (1991)).

In certain embodiments, LINGO-1 antibodies, antibody fragments,derivatives and variants thereof further comprise a targeting moiety.Targeting moieties include a protein or a peptide which directslocalization to a certain part of the body, for example, to the brain orcompartments therein. In certain embodiments, LINGO-1 antibodies,antibody fragments, derivatives and variants thereof are attached orfused to a brain targeting moiety. The brain targeting moieties areattached covalently (e.g., direct, translational fusion, or by chemicallinkage either directly or through a spacer molecule, which can beoptionally cleavable) or non-covalently attached (e.g., throughreversible interactions such as avidin, biotin, protein A, IgG, etc.).In other embodiments, the LINGO-1 antibodies, antibody fragments,derivatives and variants thereof are attached to one more braintargeting moieties. In additional embodiments, the brain targetingmoiety is attached to a plurality of LINGO-1 antibodies, antibodyfragments, derivatives and variants thereof.

A brain targeting moiety associated with a LINGO-1 antibody, antibodyfragment, derivative or variant thereof enhances brain delivery of sucha LINGO-1 antibodies, antibody fragments, derivatives and variantsthereof. A number of polypeptides have been described which, when fusedto a protein or therapeutic agent, delivers the protein or therapeuticagent through the blood brain barrier (EBB). Non-limiting examplesinclude the single domain antibody FC5 (Abuirob et al. (2005) J.Neurochem. 95, 1201-1214); mAB 83-14, a monoclonal antibody to the humaninsulin receptor (Pardridge et al. (1995) Pharmacol. Res. 12, 807-816);the B2, B6 and B8 peptides binding to the human transferrin receptor(hTfR) (Xia et al. (2000) J. Virol. 74, 11359-11366); the OX26monoclonal antibody to the transferrin receptor (Pardridge et al. (1991)J. Pharmacol. Exp. Ther. 259, 66-70); and SEQ ID NOs: 1-18 of U.S. Pat.No. 6,306,365. The contents of the above references are incorporatedherein by reference in their entirety.

Enhanced brain delivery of a LINGO-1 antibody, antibody fragment,derivative or variant thereof is determined by a number of means wellestablished in the art. For example, administering to an animal aradioactively, enzymatically or fluorescently labeled LINGO-1 antibody,antibody fragment, derivative and variant thereof linked to a braintargeting moiety; determining brain localization; and comparinglocalization with an equivalent radioactively, enzymatically orfluorescently labeled LINGO-1 antibody, antibody fragment, derivative orvariant thereof that is not associated with a brain targeting moiety.Other means of determining enhanced targeting are described in the abovereferences.

As discussed elsewhere herein, LINGO-1 antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention may befused to heterologous polypeptides to increase the in vivo half life ofthe polypeptides or for use in immunoassays using methods known in theart. For example, in one embodiment, PEG can be conjugated to theLINGO-1 antibodies of the invention to increase their half-life in vivo.Leong, S. R., et al., Cytokine 16:106 (2001); Adv. in Drug Deliv. Rev.54:531 (2002); or Weir et al., Biochem. Soc. Transactions 30:512 (2002).

Moreover, LINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be fused to marker sequences,such as a peptide to facilitate their purification or detection. Inpreferred embodiments, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

Fusion proteins can be prepared using methods that are well known in theart (see for example U.S. Pat. Nos. 5,116,964 and 5,225,538). Theprecise site at which the fusion is made may be selected empirically tooptimize the secretion or binding characteristics of the fusion protein.DNA encoding the fusion protein is then transfected into a host cell forexpression.

LINGO-1 antibodies or antigen-binding fragments, variants, orderivatives thereof of the present invention may be used innon-conjugated form or may be conjugated to at least one of a variety ofmolecules, e.g., to improve the therapeutic properties of the molecule,to facilitate target detection or for imaging or therapy of the patient.LINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be labeled or conjugated eitherbefore or after purification, when purification is performed.

In particular, LINGO-1 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention may be conjugated totherapeutic agents, prodrugs, peptides, proteins, enzymes, viruses,lipids, biological response modifiers, pharmaceutical agents, or PEG.

Those skilled in the art will appreciate that conjugates may also beassembled using a variety of techniques depending on the selected agentto be conjugated. For example, conjugates with biotin are prepared e.g.by reacting a binding polypeptide with an activated ester of biotin suchas the biotin N-hydroxysuccinimide ester. Similarly, conjugates with afluorescent marker may be prepared in the presence of a coupling agent,e.g. those listed herein, or by reaction with an isothiocyanate,preferably fluorescein-isothiocyanate. Conjugates of the LINGO-1antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention are prepared in an analogous manner.

The present invention further encompasses LINGO-1 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention conjugated to a diagnostic or therapeutic agent. The LINGO-1antibodies can be used diagnostically to, for example, monitor thedevelopment or progression of a neurological disease as part of aclinical testing procedure to, e.g., determine the efficacy of a giventreatment and/or prevention regimen. Detection can be facilitated bycoupling the LINGO-1 antibody, or antigen-binding fragment, variant, orderivative thereof to a detectable substance. Examples of detectablesubstances include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, bioluminescent materials, radioactivematerials, positron emitting metals using various positron emissiontomographies, and nonradioactive paramagnetic metal ions. See, forexample, U.S. Pat. No. 4,741,900 for metal ions which can be conjugatedto antibodies for use as diagnostics according to the present invention.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples, of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin;and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ¹¹¹Inor ⁹⁹Tc.

A LINGO-1 antibody, or antigen-binding fragment, variant, or derivativethereof also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedLINGO-1 antibody is then determined by detecting the presence ofluminescence that arises during the course of a chemical reaction.Examples of particularly useful chemiluminescent labeling compounds areluminol, isoluminol, theromatic acridinium ester, imidazole, acridiniumsalt and oxalate ester.

One of the ways in which a LINGO-1 antibody, or antigen-bindingfragment, variant, or derivative thereof can be detectably labeled is bylinking the same to an enzyme and using the linked product in an enzymeimmunoassay (EIA) (Voller, A., “The Enzyme Linked Immunosorbent Assay(ELISA)” Microbiological Associates Quarterly Publication, Walkersville,Md., Diagnostic Horizons 2:1-7 (1978)); Voller et al., J. Clin. Pathol.31:507-520 (1978); Butler, J. E., Meth. Enrymol. 73:482-523 (1981);Maggio, E. (ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla.,(1980); Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin,Tokyo (1981). The enzyme, which is bound to the LINGO-1 antibody willreact with an appropriate substrate, preferably a chromogenic substrate,in such a manner as to produce a chemical moiety which can be detected,for example, by spectrophotometric, fluorimetric or by visual means.Enzymes which can be used to detectably label the antibody include, butare not limited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. Additionally, the detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the LINGO-1antibody, or antigen-binding fragment, variant, or derivative thereof,it is possible to detect the antibody through the use of aradioimmunoassay (MA) (see, for example, Weintraub, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, (March, 1986)), which is incorporatedby reference herein). The radioactive isotope can be detected by meansincluding, but not limited to, a gamma counter, a scintillation counter,or autoradiography.

A LINGO-1 antibody, or antigen-binding fragment, variant, or derivativethereof can also be detectably labeled using fluorescence emittingmetals such as 152Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

Techniques for conjugating various moieties to a LINGO-1 antibody, orantigen-binding fragment, variant, or derivative thereof are well known,see, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting OfDrugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy,Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstromet al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2ndEd.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987);Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview”, in Monoclonal Antibodies '84: Biological And ClinicalApplications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis,Results, And Future Prospective Of The Therapeutic Use Of RadiolabeledAntibody In Cancer Therapy”, in Monoclonal Antibodies For CancerDetection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16(1985), and Thorpe et al., “The Preparation And Cytotoxic Properties OfAntibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).

VII. Expression of Antibody Polypeptides

As is well known, RNA may be isolated from the original hybridoma cellsor from other transformed cells by standard techniques, such asguanidinium isothiocyanate extraction and precipitation followed bycentrifugation or chromatography. Where desirable, mRNA may be isolatedfrom total RNA by standard techniques such as chromatography on oligo dTcellulose. Suitable techniques are familiar in the art.

In one embodiment, cDNAs that encode the light and the heavy chains ofthe antibody may be made, either simultaneously or separately, usingreverse transcriptase and DNA polymerase in accordance with well knownmethods. PCR may be initiated by consensus constant region primers or bymore specific primers based on the published heavy and light chain DNAand amino acid sequences. As discussed above, PCR also may be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries may be screened by consensus primers or largerhomologous probes, such as mouse constant region probes.

DNA, typically plasmid DNA, may be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, well known techniques set forth in detail,e.g., in the foregoing references relating to recombinant DNAtechniques. Of course, the DNA may be synthetic according to the presentinvention at any point during the isolation process or subsequentanalysis.

Following manipulation of the isolated genetic material to provideLINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention, the polynucleotides encoding theLINGO-1 antibodies are typically inserted in an expression vector forintroduction into host cells that may be used to produce the desiredquantity of LINGO-1 antibody.

Recombinant expression of an antibody, or fragment, derivative or analogthereof, e.g., a heavy or light chain of an antibody which binds to atarget molecule described herein, e.g., LINGO-1, requires constructionof an expression vector containing a polynucleotide that encodes theantibody. Once a polynucleotide encoding an antibody molecule or a heavyor light chain of an antibody, or portion thereof (preferably containingthe heavy or light chain variable domain), of the invention has beenobtained, the vector for the production of the antibody molecule may beproduced by recombinant DNA technology using techniques well known inthe art. Thus, methods for preparing a protein by expressing apolynucleotide containing an antibody encoding nucleotide sequence aredescribed herein. Methods which are well known to those skilled in theart can be used to construct expression vectors containing antibodycoding sequences and appropriate transcriptional and translationalcontrol signals. These methods include, for example, in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. The invention, thus, provides replicable vectorscomprising a nucleotide sequence encoding an antibody molecule of theinvention, or a heavy or light chain thereof, or a heavy or light chainvariable domain, operably linked to a promoter. Such vectors may includethe nucleotide sequence encoding the constant region of the antibodymolecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of theantibody may be cloned into such a vector for expression of the entireheavy or light chain.

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain is advantageouslyplaced before the heavy chain to avoid an excess of toxic free heavychain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci.USA 77:2197 (1980)). The coding sequences for the heavy and light chainsmay comprise cDNA or genomic DNA.

The term “vector” or “expression vector” is used herein to mean vectorsused in accordance with the present invention as a vehicle forintroducing into and expressing a desired gene in a host cell. As knownto those skilled in the art, such vectors may easily be selected fromthe group consisting of plasmids, phages, viruses and retroviruses. Ingeneral, vectors compatible with the instant invention will comprise aselection marker, appropriate restriction sites to facilitate cloning ofthe desired gene and the ability to enter and/or replicate in eukaryoticor prokaryotic cells.

For the purposes of this invention, numerous expression vector systemsmay be employed. For example, one class of vector utilizes DNA elementswhich are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV) or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells which have integrated the DNA into their chromosomesmay be selected by introducing one or more markers which allow selectionof transfected host cells. The marker may provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation. Additional elements may also beneeded for optimal synthesis of mRNA. These elements may include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals.

In particularly preferred embodiments the cloned variable region genesare inserted into an expression vector along with the heavy and lightchain constant region genes (preferably human) synthetic as discussedabove. In one embodiment, this is effected using a proprietaryexpression vector of Biogen IDEC, Inc., referred to as NEOSPLA (U.S.Pat. No. 6,159,730). This vector contains the cytomegaloviruspromoter/enhancer, the mouse beta globin major promoter, the SV40 originof replication, the bovine growth hormone polyadenylation sequence,neomycin phosphotransferase exon 1 and exon 2, the dihydrofolatereductase gene and leader sequence. This vector has been found to resultin very high level expression of antibodies upon incorporation ofvariable and constant region genes, transfection in CHO cells, followedby selection in G418 containing medium and methotrexate amplification.Of course, any expression vector which is capable of elicitingexpression in eukaryotic cells may be used in the present invention.Examples of suitable vectors include, but are not limited to plasmidspcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS, pRc/HCMV2, pSV40/Zeo2,pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2 (available fromInvitrogen, San Diego, Calif.), and plasmid pCI (available from Promega,Madison, Wis.). In general, screening large numbers of transformed cellsfor those which express suitably high levels if immunoglobulin heavy andlight chains is routine experimentation which can be carried out, forexample, by robotic systems. Vector systems are also taught in U.S. Pat.Nos. 5,736,137 and 5,658,570, each of which is incorporated by referencein its entirety herein. This system provides for high expression levels,e.g., >30 pg/cell/day. Other exemplary vector systems are disclosede.g., in U.S. Pat. No. 6,413,777.

In other preferred embodiments the LINGO-1 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention may be expressed using polycistronic constructs such as thosedisclosed in United States Patent Application Publication No.2003-0157641 A1, filed Nov. 18, 2002 and incorporated herein in itsentirety. In these novel expression systems, multiple gene products ofinterest such as heavy and light chains of antibodies may be producedfrom a single polycistronic construct. These systems advantageously usean internal ribosome entry site (IRES) to provide relatively high levelsof LINGO-1 antibodies, e.g., binding polypeptides, e.g.,LINGO-1-specific antibodies or immunospecific fragments thereof ineukaryotic host cells. Compatible IRES sequences are disclosed in U.S.Pat. No. 6,193,980 which is also incorporated herein. Those skilled inthe art will appreciate that such expression systems may be used toeffectively produce the full range of LINGO-1 antibodies disclosed inthe instant application.

More generally, once the vector or DNA sequence encoding a monomericsubunit of the LINGO-1 antibody has been prepared, the expression vectormay be introduced into an appropriate host cell. Introduction of theplasmid into the host cell can be accomplished by various techniqueswell known to those of skill in the art. These include, but are notlimited to, transfection (including electrophoresis andelectroporation), protoplast fusion, calcium phosphate precipitation,cell fusion with enveloped DNA, microinjection, and infection withintact virus. See, Ridgway, A. A. G. “Mammalian Expression Vectors”Vectors, Rodriguez and Denhardt, Eds., Butterworths, Boston, Mass.,Chapter 24.2, pp. 470-472 (1988). Typically, plasmid introduction intothe host is via electroporation. The host cells harboring the expressionconstruct are grown under conditions appropriate to the production ofthe light chains and heavy chains, and assayed for heavy and/or lightchain protein synthesis. Exemplary assay techniques includeenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells containing apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, operably linked to a heterologous promoter. Inpreferred embodiments for the expression of double-chained antibodies,vectors encoding both the heavy and light chains may be co-expressed inthe host cell for expression of the entire immunoglobulin molecule, asdetailed below.

As used herein, “host cells” refers to cells which harbor vectorsconstructed using recombinant DNA techniques and encoding at least oneheterologous gene. In descriptions of processes for isolation ofantibodies from recombinant hosts, the terms “cell” and “cell culture”are used interchangeably to denote the source of antibody unless it isclearly specified otherwise. In other words, recovery of polypeptidefrom the “cells” may mean either from spun down whole cells, or from thecell culture containing both the medium and the suspended cells.

A variety of host-expression vector systems may be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). Preferably, bacterial cells such asEscherichia coli, and more preferably, eukaryotic cells, especially forthe expression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese hamster ovary cells (CHO), in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

The host cell line used for protein expression is often of mammalianorigin; those skilled in the art are credited with ability topreferentially determine particular host cell lines which are bestsuited for the desired gene product to be expressed therein. Exemplaryhost cell lines include, but are not limited to, CHO (Chinese HamsterOvary), DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus), HELA(human cervical carcinoma), CV1 (monkey kidney line), COS (a derivativeof CV1 with SV40 T antigen), VERY, BHK (baby hamster kidney), MDCK, 293,WI38, R1610 (Chinese hamster fibroblast) BALBC/3T3 (mouse fibroblast),HAK (hamster kidney line), SP2/O (mouse myeloma), P3×63-Ag3.653 (mousemyeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte)and 293 (human kidney). CHO cells are particularly preferred. Host celllines are typically available from commercial services, the AmericanTissue Culture Collection or from published literature.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Nalt. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 1980) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); TIB TECH 11(5):155-215 (May, 1993); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transferand Expression, A Laboratory Manual, Stockton Press, NY (1990); and inChapters 12 and 13, Dracopoli et al. (eds), Current Prolocols in HumanGenetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol.Biol. 150:1 (1981), which are incorporated by reference herein in theirentireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Academic Press, New York, Vol. 3.(1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose or (immuno-)affinity chromatography, e.g., afterpreferential biosynthesis of a synthetic hinge region polypeptide orprior to or subsequent to the HIC chromatography step described herein.

Genes encoding LINGO-1 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention can also be expressednon-mammalian cells such as bacteria or yeast or plant cells. Bacteriawhich readily take up nucleic acids include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the heterologous polypeptides typically becomepart of inclusion bodies. The heterologous polypeptides must beisolated, purified and then assembled into functional molecules. Wheretetravalent forms of antibodies are desired, the subunits will thenself-assemble into tetravalent antibodies (WO02/096948A2).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available, e.g., Pichia pastoris.

For expression in Saccharomyces, the plasmid YRp7, for example,(Stinchcomb et al., Nature 282:39 (1979); Kingsman et al., Gene 7:141(1979); Tschemper et al., Gene 10:157 (1980)) is commonly used. Thisplasmid already contains the TRP1 gene which provides a selection markerfor a mutant strain of yeast lacking the ability to grow in tryptophan,for example ATCC No. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)). Thepresence of the trpl lesion as a characteristic of the yeast host cellgenome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.Alternatively, a preferred method for increasing the affinity ofantibodies of the invention is disclosed in US 2002 0123057 A1.

VIII. Treatment Methods Using Therapeutic LINGO-1 Antibodies

As described herein, LINGO-1 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention can relieveNgR1-mediated inhibition of axonal extension that normally takes placein CNS neurons. This is beneficial in situations where axonal extensionor neurite sprouting is needed in the brain or spinal cord. Spinal cordinjury; including partial or complete crush or severance, exemplifies asituation in which axonal extension is needed, but is normally inhibitedthrough operation of the Nogo pathway. Examples of diseases or disordersin which axonal extension and/or neurite sprouting in the brain would bebeneficial include stroke, multiple sclerosis, and otherneurodegenerative diseases or disorders such as multiple sclerosis (MS),progressive multifocal leukoencephalopathy (PML), encephalomyelitis(EPL), central pontine myelolysis (CPM), adrenoleukodystrophy,Alexander's disease, Pelizaeus Merzbacher disease (PMZ), Globoid cellLeucodystrophy (Krabbe's disease) and Wallerian Degeneration, opticneuritis, transverse myelitis, amylotrophic lateral sclerosis (ALS),Huntington's disease, Alzheimer's disease, Parkinson's disease, spinalcord injury, traumatic brain injury, post radiation injury, neurologiccomplications of chemotherapy, stroke, neuropathy, acute ischemic opticneuropathy, vitamin E deficiency, isolated vitamin E deficiencysyndrome, AR, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome,metachromatic leukodystrophy, trigeminal neuralgia, Bell's palsy, spinalcord injury and all neurological diseases related to neuronal celldeath.

The inventors have further discovered that LINGO-1 is expressed inoligodendrocytes, and contributes to oligodendrocyte biology. Solublederivatives of LINGO-1, certain polynucleotides (e.g. RNAi), as well ascertain antibodies which specifically bind to LINGO-1, as describedherein act as antagonists to LINGO-1 function in oligodendrocytes,promoting proliferation, differentiation and survival ofoligodendrocytes and promoting myelination of neurons in vitro and invivo. This is beneficial in for diseases, disorders or conditionsinvolving demyelination and dysmyelination. Examples of diseases ordisorders in which oligodendrocyte proliferation, differentiation andsurvival, and/or myelination or remyelination would be beneficialinclude multiple sclerosis (MS), progressive multifocalleukoencephalopathy (PML), encephalomyelitis (EPL), central pontinemyelolysis (CPM), adrenoleukodystrophy, Alexander's disease, PelizaeusMerzbacher disease (PMZ), Globoid cell Leucodystrophy (Krabbe'sdisease), Wallerian Degeneration, optic neuritis, transverse myelitis,amylotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer'sdisease, Parkinson's disease, spinal cord injury, traumatic braininjury, post radiation injury, neurologic complications of chemotherapy,stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolatedvitamin E deficiency syndrome, AR, Bassen-Kornzweig syndrome,Marchiafava-Bignami syndrome, metachromatic leukodystrophy, trigeminalneuralgia, and Bell's palsy.

Accordingly, one embodiment of the present invention provides methodsfor treating spinal cord injury, diseases or disorders associated withinhibition of neuronal growth in the CNS, diseases or disordersassociated with inhibition of oligodendrocyte growth or differentiation,and diseases involving demyelination or dysmyelination of CNS neurons inan animal suffering from such injury or disease or predisposed tocontract such disease, the method comprising, consisting essentially of,or consisting of administering to the animal an effective amount of aLINGO-1 antibody, or antigen-binding fragment, variant, or derivativethereof.

A therapeutic LINGO-1 antibody to be used in treatment methods disclosedherein can be prepared and used as a therapeutic agent which promotesCNS neurite outgrowth, neuronal survival, axon guidance and axonregeneration, which promotes oligodendrocyte survival, growth, and/ordifferentiation, and which promotes myelination or remyelination of CNSneurons. Characteristics of suitable therapeutic LINGO-1 antibodiesinclude: binding to LINGO-1 epitopes which result in blocking of LINGO-1activity, binding to LINGO-1 with sufficient affinity to elicit atherapeutic effect, and binding to LINGO-1 preferentially to normalbinding partners, e.g., Nogo Receptor.

Therapeutic LINGO-1 antibodies may be monoclonal, chimeric or humanizedantibodies, or fragments of antibodies that bind specifically toLINGO-1. The antibodies may be monovalent, bivalent, polyvalent, orbifunctional antibodies. Antibody fragments include without limitationFab F(ab′)₂, and Fv fragments.

Therapeutic LINGO-1 antibodies, or antigen-binding fragments, variantsor derivatives thereof according to the invention can be used inunlabeled or unconjugated form, or can be coupled or linked to drugs,labels or stabilization agents which may or may not exert additionaltherapeutic effects.

A specific dosage and treatment regimen for any particular patient willdepend upon a variety of factors, including the particular LINGO-1antibody, or antigen-binding fragment, variant or derivative thereofused, the patient's age, body weight, general health, sex, and diet, andthe time of administration, rate of excretion, drug combination, and theseverity of the particular disease being treated. Judgment of suchfactors by medical caregivers is within the ordinary skill in the art.The amount will also depend on the individual patient to be treated, theroute of administration, the type of formulation, the characteristics ofthe compound used, the severity of the disease, and the desired effect.The amount used can be determined by pharmacological and pharmacokineticprinciples well known in the art.

In the methods of the invention the LINGO-1 antibodies, orantigen-binding fragments, variants or derivatives thereof may beadministered directly to the nervous system, intracerebroventricularly,or intrathecally, e.g. into a chronic lesion of MS, as discussed in moredetail below.

In various embodiments, a LINGO-1 antibody as described above is anantagonist of LINGO-1 activity. In certain embodiments, for example,binding of an antagonist LINGO-1 antibody to LINGO-1, as expressed onneurons, blocks myelin-associated neurite outgrowth inhibition orneuronal cell death. In other embodiments, binding of the LINGO-1antibody to LINGO-1, as expressed on oligodendrocytes, blocks inhibitionof oligodendrocyte growth or differentiation, or blocks demyelination ordysmyelination of CNS neurons.

In methods of the present invention, a LINGO-1 antibody, or anantigen-binding fragment, variant, or derivative thereof, in particularthe LINGO-1 antibodies described herein, can be administered directly asa preformed polypeptide, or indirectly through a nucleic acid vector, topermit beneficial axonal outgrowth, promote oligodendrocyteproliferation, differentiation, and survival, and/or promote myelinationor remyelination.

In certain embodiments, a subject may be treated with a nucleic acidmolecule encoding a LINGO-1 antibody, or antigen-binding fragmentvariant, or analog thereof, e.g., in a vector. Doses for nucleic acidsencoding polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1μg to 10 mg, or 30-300 μg DNA per patient. Doses for infectious viralvectors vary from 10-100, or more, virions per dose.

In some embodiments of the present invention a LINGO-1 antibody, or anantigen-binding fragment, variant, or derivative thereof is administeredin a treatment method that includes: (1) transforming or transfecting animplantable host cell with a nucleic acid, e.g., a vector, thatexpresses a LINGO-1 antibody, or an antigen-binding fragment, variant,or derivative thereof; and (2) implanting the transformed host cell intoa mammal, at the site of a disease, disorder or injury. For example, thetransformed host cell can be implanted at the site of a spinal cordinjury or at a site of dysmyelination. In some embodiments of theinvention, the implantable host cell is removed from a mammal,temporarily cultured, transformed or transfected with an isolatednucleic acid encoding a a LINGO-1 antibody, and implanted back into thesame mammal from which it was removed. The cell can be, but is notrequired to be, removed from the same site at which it is implanted.Such embodiments, sometimes known as ex vivo gene therapy, can provide acontinuous supply of the LINGO-1 polypeptide, localized at the site ofsite of action, for a limited period of time.

The methods for treating spinal cord injury, diseases or disordersassociated with inhibition of neuronal growth in the CNS, diseases ordisorders associated with inhibition of oligodendrocyte growth ordifferentiation, and diseases involving demyelination or dysmyelinationof CNS neurons comprising administration of a LINGO-1 antibody, orantigen-binding fragment, variant, or derivative thereof of theinvention are typically tested in vitro, and then in vivo in anacceptable animal model, for the desired therapeutic or prophylacticactivity, prior to use in humans. Suitable animal models, includingtransgenic animals, are will known to those of ordinary skill in theart. For example, in vitro assays to demonstrate the therapeutic utilityof LINGO-1 antibody described herein include the effect of a LINGO-1antibody on a cell line or a patient tissue sample. The effect of theLINGO-1 antibody on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art, such as theassays disclosed elsewhere herein. In accordance with the invention, invitro assays which can be used to determine whether administration of aspecific LINGO-1 antibody is indicated, include in vitro cell cultureassays in which a patient tissue sample is grown in culture, and exposedto or otherwise administered a compound, and the effect of such compoundupon the tissue sample is observed.

Supplementary active compounds also can be incorporated into thecompositions of the invention. For example, a LINGO-1 antibody, orantigen-binding fragment, variant, or derivative thereof of theinvention may be coformulated with and/or coadministered with one ormore additional therapeutic agents.

The invention encompasses any suitable delivery method for a LINGO-1antibody, or antigen-binding fragment, variant, or derivative thereof ofthe invention to a selected target tissue, including bolus injection ofan aqueous solution or implantation of a controlled-release system. Useof a controlled-release implant reduces the need for repeat injections.

IX. Pharmaceutical Compositions and Administration Methods

Methods of preparing and administering LINGO-1 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention to a subject in need thereof are well known to or are readilydetermined by those skilled in the art. The route of administration ofthe LINGO-1 antibody, or antigen-binding fragment, variant, orderivative thereof may be, for example, oral, parenteral, by inhalationor topical. The term parenteral as used herein includes, e.g.,intravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, rectal or vaginal administration. While all these forms ofadministration are clearly contemplated as being within the scope of theinvention, a form for administration would be a solution for injection,in particular for intravenous or intraarterial injection or drip.Usually, a suitable pharmaceutical composition for injection maycomprise a buffer (e.g. acetate, phosphate or citrate buffer), asurfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. humanalbumin), etc. However, in other methods compatible with the teachingsherein, LINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be delivered directly to thesite of the adverse cellular population thereby increasing the exposureof the diseased tissue to the therapeutic agent.

As previously discussed, LINGO-1 antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention may beadministered in a pharmaceutically effective amount for the in vivotreatment of mammalian spinal cord injury, diseases or disordersassociated with inhibition of neuronal growth in the CNS, diseases ordisorders associated with inhibition of oligodendrocyte growth ordifferentiation, and diseases involving demyelination or dysmyelinationof CNS. In this regard, it will be appreciated that the disclosedantibodies will be formulated so as to facilitate administration andpromote stability of the active agent. Preferably, pharmaceuticalcompositions in accordance with the present invention comprise apharmaceutically acceptable, non-toxic, sterile carrier such asphysiological saline, non-toxic buffers, preservatives and the like. Forthe purposes of the instant application, a pharmaceutically effectiveamount of a LINGO-1 antibody, or antigen-binding fragment, variant, orderivative thereof, conjugated or unconjugated, shall be held to mean anamount sufficient to achieve effective binding to a target and toachieve a benefit, e.g., to ameliorate symptoms of a disease or disorderor to detect a substance or a cell.

The pharmaceutical compositions used in this invention comprisepharmaceutically acceptable carriers, including, e.g., ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

Preparations for parenteral administration includes sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the subject invention, pharmaceutically acceptable carriersinclude, but are not limited to, 0.01-0.1M and preferably 0.05Mphosphate buffer or 0.8% saline. Other common parenteral vehiclesinclude sodium phosphate solutions, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's, or fixed oils. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers, suchas those based on Ringer's dextrose, and the like. Preservatives andother additives may also be present such as for example, antimicrobials,antioxidants, chelating agents, and inert gases and the like.

More particularly, pharmaceutical compositions suitable for injectableuse include sterile aqueous solutions (where water soluble) ordispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersions. In such cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It should be stable under the conditions ofmanufacture and storage and will preferably be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Suitableformulations for use in the therapeutic methods disclosed herein aredescribed in Remington's Pharmaceutical Sciences, Mack Publishing Co.,16th ed. (1980).

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., a LINGO-1 antibody, orantigen-binding fragment, variant, or derivative thereof, by itself orin combination with other active agents) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedherein, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle, which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying,which yields a powder of an active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The preparations for injections are processed, filled into containerssuch as ampoules, bags, bottles, syringes or vials, and sealed underaseptic conditions according to methods known in the art. Further, thepreparations may be packaged and sold in the form of a kit such as thosedescribed in co-pending U.S. Ser. No. 09/259,337 (US-2002-0102208 A1),which is incorporated herein by reference in its entirety. Such articlesof manufacture will preferably have labels or package inserts indicatingthat the associated compositions are useful for treating a subjectsuffering from, or predisposed to autoimmune or neoplastic disorders.

Parenteral formulations may be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionsmay be administered at specific fixed or variable intervals, e.g., oncea day, or on an “as needed” basis.

Certain pharmaceutical compositions used in this invention may be orallyadministered in an acceptable dosage form including, e.g., capsules,tablets, aqueous suspensions or solutions. Certain pharmaceuticalcompositions also may be administered by nasal aerosol or inhalation.Such compositions may be prepared as solutions in saline, employingbenzyl alcohol or other suitable preservatives, absorption promoters toenhance bioavailability, and/or other conventional solubilizing ordispersing agents.

The amount of a LINGO-1 antibody, or fragment, variant, or derivativethereof that may be combined with the carrier materials to produce asingle dosage form will vary depending upon the host treated and theparticular mode of administration. The composition may be administeredas a single dose, multiple doses or over an established period of timein an infusion. Dosage regimens also may be adjusted to provide theoptimum desired response (e.g., a therapeutic or prophylactic response).

In keeping with the scope of the present disclosure, LINGO-1 antibodies,or antigen-binding fragments, variants, or derivatives thereof of theinvention may be administered to a human or other animal in accordancewith the aforementioned methods of treatment in an amount sufficient toproduce a therapeutic effect. The LINGO-1 antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention can beadministered to such human or other animal in a conventional dosage formprepared by combining the antibody of the invention with a conventionalpharmaceutically acceptable carrier or diluent according to knowntechniques. It will be recognized by one of skill in the art that theform and character of the pharmaceutically acceptable carrier or diluentis dictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.Those skilled in the art will further appreciate that a cocktailcomprising one or more species of LINGO-1 antibodies, or antigen-bindingfragments, variants, or derivatives thereof of the invention may proveto be particularly effective.

Effective doses of the compositions of the present invention, fortreatment of spinal cord injury, diseases or disorders associated withinhibition of neuronal growth in the CNS, diseases or disordersassociated with inhibition of oligodendrocyte growth or differentiation,and diseases involving demyelination or dysmyelination of CNS varydepending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic. Usually, the patientis a human but non-human mammals including transgenic mammals can alsobe treated. Treatment dosages may be titrated using routine methodsknown to those of skill in the art to optimize safety and efficacy.

For treatment of spinal cord injury, diseases or disorders associatedwith inhibition of neuronal growth in the CNS, diseases or disordersassociated with inhibition of oligodendrocyte growth or differentiation,and diseases involving demyelination or dysmyelination of CNS with aLINGO-1 antibody, or antigen-binding fragment, variant, or derivativethereof, the dosage can range, e.g., from about 0.0001 to 100 mg/kg, andmore usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg,0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body weight. Forexample dosages can be 1 mg/kg body weight or 10 mg/kg body weight orwithin the range of 1-10 mg/kg, preferably at least 1 mg/kg. Dosesintermediate in the above ranges are also intended to be within thescope of the invention. Subjects can be administered such doses daily,on alternative days, weekly or according to any other scheduledetermined by empirical analysis. An exemplary treatment entailsadministration in multiple dosages over a prolonged period, for example,of at least six months. Additional exemplary treatment regimes entailadministration once per every two weeks or once a month or once every 3to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15 mg/kgon consecutive days, 30 mg/kg on, alternate days or 60 mg/kg weekly. Insome methods, two or more monoclonal antibodies with different bindingspecificities are administered simultaneously, in which case the dosageof each antibody administered falls within the ranges indicated.

LINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can be administered on multipleoccasions. Intervals between single dosages can be daily, weekly,monthly or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of target polypeptide or target molecule in thepatient. In some methods, dosage is adjusted to achieve a plasmapolypeptide concentration of 1-1000 μg/ml and in some methods 25-300μg/ml. Alternatively, LINGO-1 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention can be administered asa sustained release formulation, in which case less frequentadministration is required. Dosage and frequency vary depending on thehalf-life of the antibody in the patient. The half-life of a LINGO-1antibody can also be prolonged via fusion to a stable polypeptide ormoeity, e.g., albumin or PEG. In general, humanized antibodies show thelongest half-life, followed by chimeric antibodies and nonhumanantibodies. In one embodiment, the LINGO-1 antibodies, orantigen-binding fragments, variants, or derivatives thereof of theinvention can be administered in unconjugated form, In anotherembodiment, the LINGO-1 antibodies, or antigen-binding fragments,variants, or derivatives thereof of the invention can be administeredmultiple times in conjugated form. In still another embodiment, LINGO-1antibodies, or antigen-binding fragments, variants, or derivativesthereof of the invention can be administered in unconjugated form, thenin conjugated form, or vice versa.

The compositions of the present invention may be administered by anysuitable method, e.g., parenterally, intraventricularly, orally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, infrasternal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques. As describedpreviously, LINGO-1 antibodies, or antigen-binding fragments, variants,or derivatives thereof of the invention act in the nervous system topromote survival, proliferation and differentiation of oligodendrocytesand myelination of neurons and neuronal survival, axon regeneration andaxon guidance. Accordingly, in the methods of the invention, the LINGO-1antibodies, or antigen-binding fragments, variants, or derivativesthereof are administered in such a way that they cross the blood-brainbarrier. This crossing can result from the physico-chemical propertiesinherent in the LINGO-1 antibody molecule itself, from other componentsin a pharmaceutical formulation, or from the use of a mechanical devicesuch as a needle, cannula or surgical instruments to breach theblood-brain barrier. Where the LINGO-1 antibody is a molecule that doesnot inherently cross the blood-brain barrier, e.g., a fusion to a moietythat facilitates the crossing, suitable routes of administration are,e.g., intrathecal or intracranial, e.g., directly into a chronic lesionof MS. Where the LINGO-1 antibody is a molecule that inherently crossesthe blood-brain barrier, the route of administration may be by one ormore of the various routes described below. In some methods, antibodiesare administered as a sustained release composition or device, such as aMedipad™ device. Delivery across the blood brain barrier can be enhancedby a carrying molecule, such as anti-Fc receptor, transferrin,anti-insulin receptor or a toxin conjugate or penetration enhancer.

The LINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof used in the methods of the invention may be directlyinfused into the brain. Various implants for direct brain infusion ofcompounds are known and are effective in the delivery of therapeuticcompounds to human patients suffering from neurological disorders. Theseinclude chronic infusion into the brain using a pump, stereotacticallyimplanted, temporary interstitial catheters, permanent intracranialcatheter implants, and surgically implanted biodegradable implants. See,e.g., Gill et al., “Direct brain infusion of glial cell line-derivedneurotrophic factor in Parkinson disease,” Nature Med. 9: 589-95 (2003);Scharfen et al., “High Activity Iodine-125 Interstitial Implant ForGliomas,” Int. J. Radiation Oncology Biol. Phys. 24(4):583-91 (1992);Gaspar et al., “Permanent ¹²⁵I Implants for Recurrent MalignantGliomas,” Int. J. Radiation Oncology Biol. Phys. 43(5):977-82 (1999);chapter 66, pages 577-580, Bellezza et al., “Stereotactic InterstitialBrachytherapy,” in Gildenberg et al., Textbook of Stereotactic andFunctional Neurosurgery, McGraw-Hill (1998); and Brem et al., “TheSafety of Interstitial Chemotherapy with BCNU-Loaded Polymer Followed byRadiation Therapy in the Treatment of Newly Diagnosed Malignant Gliomas:Phase I Trial,” J. Neuro-Oncology 26:111-23 (1995).

The compositions may also comprise a LINGO-1 antibody dispersed in abiocompatible carrier material that functions as a suitable delivery orsupport system for the compounds. Suitable examples of sustained releasecarriers include semipermeable polymer matrices in the form of shapedarticles such as suppositories or capsules. Implantable or microcapsularsustained release matrices include polylactides (U.S. Pat. No.3,773,319; EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-56 (1985));poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate (Langer etal., J. Biomed. Mater. Res. 15:167-277 (1981); Langer, Chem. Tech.12:98-105 (1982)) or poly-D-(−)-3hydroxybutyric acid (EP 133,988).

In some embodiments of the invention, a LINGO-1 antibody, orantigen-binding fragment, variant, or derivative thereof of theinvention is administered to a patient by direct infusion into anappropriate region of the brain. See, e.g., Gill et al., supra.Alternative techniques are available and may be applied to administer aLINGO-1 antibody according to the invention. For example, stereotacticplacement of a catheter or implant can be accomplished using theRiechert-Mundinger unit and the ZD (Zamorano-Dujovny) multipurposelocalizing unit. A contrast-enhanced computerized tomography (CT) scan,injecting 120 ml of omnipaque, 350 mg iodine/ml, with 2 mm slicethickness can allow three-dimensional multiplanar treatment planning(STP, Fischer, Freiburg, Germany). This equipment permits planning onthe basis of magnetic resonance imaging studies, merging the CT and MRItarget information for clear target confirmation.

The Leksell stereotactic system (Downs Surgical, Inc., Decatur, Ga.)modified for use with a GE CT scanner (General Electric Company,Milwaukee, Wis.) as well as the Brown-Roberts-Wells (BRW) stereotacticsystem (Radionics, Burlington, Mass.) can be used for this purpose.Thus, on the morning of the implant, the annular base ring of the BRWstereotactic frame can be attached to the patient's skull. Serial CTsections can be obtained at 3 mm intervals though the (target tissue)region with a graphite rod localizer frame clamped to the base plate. Acomputerized treatment planning program can be run on a VAX 11/780computer (Digital Equipment Corporation, Maynard, Mass.) using CTcoordinates of the graphite rod images to map between CT space and BRWspace.

LINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention can optionally be administered incombination with other agents that are effective in treating thedisorder or condition in need of treatment (e.g., prophylactic ortherapeutic).

X. Diagnostics

The invention further provides a diagnostic method useful duringdiagnosis of neronal disorders or injuries, which involves measuring theexpression level of LINGO-1 protein or transcript in tissue or othercells or body fluid from an individual and comparing the measuredexpression level with a standard LINGO-1 expression levels in normaltissue or body fluid, whereby an increase in the expression levelcompared to the standard is indicative of a disorder.

LINGO-1-specific antibodies can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting proteinexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA), immunoprecipitation, or western blotting. Suitable assaysare described in more detail elsewhere herein.

By “assaying the expression level of LINGO-1 polypeptide” is intendedqualitatively or quantitatively measuring or estimating the level ofLINGO-1 polypeptide in a first biological sample either directly (e.g.,by determining or estimating absolute protein level) or relatively(e.g., by comparing to the cancer associated polypeptide level in asecond biological sample). Preferably, LINGO-1 polypeptide expressionlevel in the first biological sample is measured or estimated andcompared to a standard LINGO-1 polypeptide level, the standard beingtaken from a second biological sample obtained from an individual nothaving the disorder or being determined by averaging levels from apopulation of individuals not having the disorder. As will beappreciated in the art, once the “standard” LINGO-1 polypeptide level isknown, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, cell line, tissue culture, or other source of cellspotentially expressing LINGO-1. Methods for obtaining tissue biopsiesand body fluids from mammals are well known in the art.

LINGO-1 antibodies for use in the diagnostic methods described aboveinclude any LINGO-1 antibody which specifically binds to a LINGO-1 geneproduct, as described elsewhere herein.

XI. Immunoassays

LINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., eds,Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NewYork, Vol. 1 (1994), which is incorporated by reference herein in itsentirety). Exemplary immunoassays are described briefly below (but arenot intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C. washing the beads in lysis buffer and resuspendingthe beads in SDS/sample buffer. The ability of the antibody of interestto immunoprecipitate a particular antigen can be assessed by, e.g.,western blot analysis. One of skill in the art would be knowledgeable asto the parameters that can be modified to increase the binding of theantibody to an antigen and decrease the background (e.g., pre-clearingthe cell lysate with sepharose beads). For further discussion regardingimmunoprecipitation protocols see, e.g., Ausubel et al., eds, CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., New York, Vol.1 (1994) at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al., eds, Current Protocols in Molecular Biology, John Wiley & Sons,Inc., New York Vol. 1 (1994) at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al., eds, Current Protocols in Molecular Biology, John Wiley& Sons, Inc., New York, Vol. 1 (1994) at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest is conjugated to a labeled compound (e.g., ³H or¹²⁵I) in the presence of increasing amounts of an unlabeled secondantibody.

LINGO-1 antibodies, or antigen-binding fragments, variants, orderivatives thereof of the invention, additionally, be employedhistologically, as in immunofluorescence, immunoelectron microscopy ornon-immunological assays, for in situ detection of cancer antigen geneproducts or conserved variants or peptide fragments thereof. In situdetection may be accomplished by removing a histological specimen from apatient, and applying thereto a labeled LINGO-1 antibody, orantigen-binding fragment, variant, or derivative thereof, preferablyapplied by overlaying the labeled antibody (or fragment) onto abiological sample. Through the use of such a procedure, it is possibleto determine not only the presence of LINGO-1 protein, or conservedvariants or peptide fragments, but also its distribution in the examinedtissue. Using the present invention, those of ordinary skill willreadily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

Immunoassays and non-immunoassays for LINGO-1 gene products or conservedvariants or peptide fragments thereof will typically comprise incubatinga sample, such as a biological fluid, a tissue extract, freshlyharvested cells, or lysates of cells which have been incubated in cellculture, in the presence of a detectably labeled antibody capable ofbinding to LINGO-1 or conserved variants or peptide fragments thereof,and detecting the bound antibody by any of a number of techniqueswell-known in the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled LINGO-1 antibody, orantigen-binding fragment, variant, or derivative thereof. The solidphase support may then be washed with the buffer a second time to removeunbound antibody. Optionally the antibody is subsequently labeled. Theamount of bound label on solid support may then be detected byconventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of LINGO-1 antibody, orantigen-binding fragment, variant, or derivative thereof may bedetermined according to well known methods. Those skilled in the artwill be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

There are a variety of methods available for measuring the affinity ofan antibody-antigen interaction, but relatively few for determining rateconstants. Most of the methods rely on either labeling antibody orantigen, which inevitably complicates routine measurements andintroduces uncertainties in the measured quantities.

Surface plasmon reasonance (SPR) as performed on BIAcore offers a numberof advantages over conventional methods of measuring the affinity ofantibody-antigen interactions: (i) no requirement to label eitherantibody or antigen; (ii) antibodies do not need to be purified inadvance, cell culture supernatant can be used directly; (iii) real-timemeasurements, allowing rapid semi-quantitative comparison of differentmonoclonal antibody interactions, are enabled and are sufficient formany evaluation purposes; (iv) biospecific surface can be regenerated sothat a series of different monoclonal antibodies can easily be comparedunder identical conditions; (v) analytical procedures are fullyautomated, and extensive series of measurements can be performed withoutuser intervention. BIAapplications Handbook, version AB (reprinted1998), BIACORE code No. BR-1001-86; BIAtechnology Handbook, version AB(reprinted 1998), BIACORE code No. BR-1001-84.

SPR based binding studies require that one member of a binding pair beimmobilized on a sensor surface. The binding partner immobilized isreferred to as the ligand. The binding partner in solution is referredto as the analyte. In some cases, the ligand is attached indirectly tothe surface through binding to another immobilized molecule, which isreferred as the capturing molecule. SPR response reflects a change inmass concentration at the detector surface as analytes bind ordissociate.

Based on SPR, real-time BIAcore measurements monitor interactionsdirectly as they happen. The technique is well suited to determinationof kinetic parameters. Comparative affinity ranking is extremely simpleto perform, and both kinetic and affinity constants can be derived fromthe sensorgram data.

When analyte is injected in a discrete pulse across a ligand surface,the resulting sensorgram can be divided into three essential phases: (i)Association of analyte with ligand during sample injection; (ii)Equilibrium or steady state during sample injection, where the rate ofanalyte binding is balanced by dissociation from the complex; (iii)Dissociation of analyte from the surface during buffer flow.

The association and dissociation phases provide information on thekinetics of analyte-ligand interaction (k_(a) and k_(d), the rates ofcomplex formation and dissociation, k_(d)/k_(a)=K_(D)). The equilibriumphase provides information on the affinity of the analyte-ligandinteraction (K_(D)).

BIAevaluation software provides comprehensive facilities for curvefitting using both numerical integration and global fitting algorithms.With suitable analysis of the data, separate rate and affinity constantsfor interaction can be obtained from simple BIAcore investigations. Therange of affinities measurable by this technique is very broad rangingfrom mM to pM.

Epitope specificity is an important characteristic of a monoclonalantibody. Epitope mapping with BIAcore, in contrast to conventionaltechniques using radioimmunoassay, ELISA or other surface adsorptionmethods, does not require labeling or purified antibodies, and allowsmulti-site specificity tests using a sequence of several monoclonalantibodies. Additionally, large numbers of analyses can be processedautomatically.

Pair-wise binding experiments test the ability of two Abs to bindsimultaneously to the same antigen. MAbs directed against separateepitopes will bind independently, whereas MAbs directed againstidentical or closely related epitopes will interfere with each other'sbinding. These binding experiments with BIAcore are straightforward tocarry out.

For example, one can use a capture molecule to bind the first Mab,followed by addition of antigen and second MAb sequentially. Thesensorgrams will reveal: 1. how much of the antigen binds to first Mab,2. to what extent the second MAb binds to the surface-attached antigen,3. if the second MAb does not bind, whether reversing the order of thepair-wise test alters the results.

Peptide inhibition is another technique used for epitope mapping. Thismethod can complement pair-wise antibody binding studies, and can relatefunctional epitopes to structural features when the primary sequence ofthe antigen is known. Peptides or antigen fragments are tested forinhibition of binding of different MAbs to immobilized antigen. Peptideswhich interfere with binding of a given MAb are assumed to bestructurally related to the epitope defined by that MAb.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2nd Ed., Sambrook et al., ed., Cold SpringHarbor Laboratory Press: (1989); Molecular Cloning: A Laboratory Manual,Sambrook et al., ed., Cold Springs Harbor Laboratory, New York (1992),DNA Cloning, D. N. Glover ed., Volumes I and II (1985); OligonucleotideSynthesis, M. J. Gait ed., (1984); Mullis et al. U.S. Pat. No.4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds.(1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds.(1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc.,(1987); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, APractical Guide To Molecular Cloning (1984); the treatise, Methods InEnzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors ForMammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring HarborLaboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al.eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer andWalker, eds., Academic Press, London (1987); Handbook Of ExperimentalImmunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., (1986);Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., (1986); and in Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).

General principles of antibody engineering are set forth in AntibodyEngineering, 2nd edition, C. A. K. Borrebaeck, Ed., Oxford Univ. Press(1995). General principles of protein engineering are set forth inProtein Engineering, A Practical Approach, Rickwood, D., et al., Eds.,IRL Press at Oxford Univ. Press, Oxford, Eng. (1995). General principlesof antibodies and antibody-hapten binding are set forth in: Nisonoff,A., Molecular Immunology, 2nd ed., Sinauer Associates, Sunderland, Mass.(1984); and Steward, M. W., Antibodies, Their Structure and Function,Chapman and Hall, New York, N.Y. (1984). Additionally, standard methodsin immunology known in the art and not specifically described aregenerally followed as in Current Protocols in Immunology, John Wiley &Sons, New York; Stites et al. (eds), Basic and Clinical-Immunology (8thed.), Appleton & Lange, Norwalk, Conn. (1994) and Mishell and Shiigi(eds), Selected Methods in Cellular Immunology, W.H. Freeman and Co.,New York (1980).

Standard reference works setting forth general principles of immunologyinclude Current Protocols in Immunology, John Wiley & Sons, New York;Klein, J. Immunology: The Science of Self-Nonself Discrimination, JohnWiley & Sons, New York (1982); Kennett, R., et al., eds., MonoclonalAntibodies, Hybridoma: A New Dimension in Biological Analyses, PlenumPress, New York (1980); Campbell, A., “Monoclonal Antibody Technology”in Burden, R., et al., eds., Laboratory Techniques in Biochemistry andMolecular Biology, Vol. 13, Elsevere, Amsterdam (1984), Kuby Immunnology4^(th) ed. Ed. Richard A. Goldsby, Thomas J. Kindt and Barbara A.Osborne, H. Freemand & Co. (2000); Roitt, I., Brostoff, J. and Male D.,Immunology 6^(th) ed. London: Mosby (2001); Abbas A., Abul, A. andLichtman, A., Cellular and Molecular Immunology Ed. 5, Elsevier HealthSciences Division (2005); Kontermann and Dubel, Antibody Engineering,Springer Verlan (2001); Sambrook and Russell, Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Press (2001); Lewin, Genes VIII,Prentice Hall (2003); Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Press (1988); Dieffenbach and Dveksler, PCR PrimerCold Spring Harbor Press (2003).

All of the references cited above, as well as all references citedherein, are incorporated herein by reference in their entireties.

EXAMPLES Example 1

Identification of Anti-LINGO-1 Antibodies by Phage Display

Li13 and Li33 were identified as Fab-phages that specifically bound toLINGO-1 using phage display as described in PCT/US2006/026271, filedJul. 7, 2006, which is herein incorporated by reference in its entirety.Li81 is derived from Li13 and Li33. It includes the Li13 light chain andan affinity matured heavy chain. The isolation of Li81 is described inmore detail in PCT/US2008/000316, filed Jan. 9, 2008, which isincorporated herein by reference in its entirety. An aglycosylated fullyhuman monoclonal antibody was created from the Li81 Fab, Li81 (agly),and its production is also detailed in PCT/US2008/000316. Li62 isderived from Li33. It, includes the Li33 heavy chain and a light chainthat was identified in a library screen.

The isolation of the Fab fragments is summarized briefly as follows. Fabfragments were isolated from phage display libraries as described inHoet et al., Nat. Biotech. 23:344-348 (2005); Rauchenberger, et al., J.Biol. Chem. 278:194-205 (2003); and Knappik, et al., J. Mol. Biol.296:57-86 (2000), all of which are incorporated herein by reference intheir entireties.

Li62 and Li81 Fabs, as well as Li62 (agly) and Li81(agly), have beenpurified and demonstrated to bind to specifically to LINGO-1 by bothELISA and FACS. Assays were performed as described in PCT/US2008/000316.

Example 2 Li62 and Li81 Promote Myelination In Vitro

The role of Li62 and Li81 in myelination was investigated in vitro bytreating co-cultures of dorsal root ganglion (DRG) neurons andoligodendrocytes with Li62 (agly) and Li81 (agly). The DRG neurons werethen tested for myelination using Western blotting. For these studies,it was necessary to first generate primary cultures of DRG neurons andof oligodendrocytes.

Female Long Evans rat E14-E17 embryonic dorsal root ganglia werecultured as described by Plant et al., J. Neurosci. 22:6083-91 (2002).Dissected DRGs were plated on poly-L-lysine-coated cover slips (100μg/ml) for 2 weeks. The cells were incubated in the presence offluorodeoxyuridine for days 2-6 and in NLA medium containing 1×B27, 100ng/ml NGF (Gibco) for days 8-11.

Female Long Evans post-natal day 2 (P2) rat oligodendrocytes werecultured as described by Conn, Meth. Neurosci. 2:1-4 (Academic Press;1990) with modifications as follows. Briefly, the forebrain wasextirpated from P2 rats and placed in cold HBSS medium (Gibco). Thetissue fragments were cut into 1 mm pieces and incubated at 37° C. for15 min in 0.01% trypsin and 10 μg/ml DNase. Dissociated cells wereplated on a poly-L-lysine coated T75 tissue culture flasks and grown inDMEM with 20% fetal bovine serum at 37° C. for 10 days. A2B5-positiveoligodendrocytes were collected by shaking the flasks overnight at 200rpm at 37° C. The A2B5 oligodendrocytes were cultured for 7 days in DMEM(Gibco) containing 25 mM D-glucose, 4 mM L-glutamine, 1 mM sodiumpyruvate, 50 μg/ml human apo-transferrin, 5 μg/ml bovine pancreaticinsulin, 30 nM sodium selenate, 10 nM hydrocortisone, 10 nM D-biotin, 1mg/ml BSA, 10 ng/ml FGF and PDGF (Peprotech). The cells were thenharvested by trypsinization. The cells then co-cultured with the DRGneurons in the presence or absence of 1.0, 0.30, 0.10, or 0.03 μg/ml ofLi62 (agly) or Li81 (agly), or a negative control antibody (h5C8 Ctrl)in NLA medium containing 2% fetal bovine serum, 50 μg/ml ascorbic acid,100 ng/ml NGF (Gibco). One of skill in the art would be able todetermine an effective dose using assays described herein.

The culture medium was changed and the antibodies or antibody fragmentswere replenished every three days. After 3 weeks at 37° C., theco-cultured cells were lysed and subjected to Western blot analysis toquantify the MBP and MOG (FIG. 1). Based on Western blot analyses,co-cultured cells treated with Li62 (agly) and Li81 (agly) showedincreased levels of both MBP and MOG compared to control-antibodytreated co-cultures. Similar results were obtained using Li62 and Li81Fabs. These data suggest that both Li62 and Li81 can promote myelinationin vitro and can promote mature oligodendrocyte axon interactions andmyelination compared to control-antibody treated co-cultures.

Example 3 Li62 and Li81 Variants

In order to identify antibodies with improved affinity, Li62 and Li81variants were isolated by targeted phage display. The variants includedalterations in the amino acid sequence of the VH CDR3 sequence in eachof the Fabs. Eighteen Li62 variants had improved affinities as shownbelow in Table 5.

TABLE 5 Li62 Variants Fold- Li62 Heavy Chain Li62 Improve- SEQCDR3 Sequence ELISA Variant ment ID NO E G H N D W Y F D L Signal RepsB06 8 17 Y Y Q 3 B12 8 18 Q Y V 3 F06 12 19 D Y 3 12 B01 8 20 Q Y 3 D0915 21 A D I F 3 D12 8 22 Y 3 26 F01 9 23 R Y P 3 F02 8 24 D Y 3 F06 8 25R Y 3 2 F10 5 26 S I R 3 G08 10 27 Q Y V 3 4 H08 6 28 Y N G 0.5 C10 1129 Y Y 3 4 C02 8 30 T Y L 3 D05 10 31 Y Y E 3 2 F02 16 32 L I F Q 3 C109 33 Q F 3 H08 9 34 T Y 3

Additionally, fifteen Li81 variants had improved affinities as shownbelow in Table 6.

TABLE 6 Li81 Variants Fold- Li81 Heavy Chain Li81 Improve- SEQCDR3 Sequence ELISA variant ment ID NO E G D N D A F D I Signal Reps F099 35 E V 2.5 G02 6 36 Y T 3 H03 9 37 T 3 A12 15.1 38 S 2.6 C02 6 39 T2.8 2 C11 15.1 40 Y R 2 D11 6 41 V S 2.1 E05 15 42 D V M 2.9 H04 6 43 YF 3 B04 8 44 D Y M 3 A02 8 45 Q Y T Y L 3 B12 6 46 D T 3 H06 6 47 A D 3H08 6 48 E M 3 E07 6 49 E Y T Y 3

Example 4 Li81 Promotes Rat Oligodendrocyte Differentiation

The ability of Li81 to promote the differentiation of rat A2B5+progenitor cells into mature MBP+ myelinating oligodendroctyes wastested. This process was studied in vitro by plating primary ratforebrain A2B5+ cells into 24-well culture plates, treating cultures for72 hr with Li81 (agly), and staining cultures for myelin basic protein(MBP) expression by Western blotting. In Western blots, myelinoligodendrocyte glycoprotein (MOG) expression was also used as a markerfor maturation.

Treatment with Li81 (agly) resulted in more highly differentiated,mature oligodendrocytes as evidenced by increases in the length of cellprocesses and the presence of abundant myelin sheet structures that arestained by the anti-MBP antibody. A dose-dependent increase in number ofmature oligodendrocytes was observed. The lowest concentration of Li81(agly) with a detectable effect on MBP production was 0.1 μg/mL. A smallpercentage of less differentiated oligodendrocytes was seen in thecontrol antibody treated cells. By Western blotting, there was adose-dependent increase in MBP and MOG expression in the Li81 (agly)treated samples (FIG. 2). No expression was observed with the isotypecontrol antibody at any concentration. The complex pattern of MBP bandsresults from alternatively spliced forms of the MBP protein. Similarresults were obtained using Li81 Fab. These results indicate that Li81can promote differentiation of rat A2B5+ progenitor cells into matureMBP+ myelinating oligodendroctyes in vitro.

Example 5 Li81 Promotes Human Oligodendrocyte Differentiation

The ability of Li81 to promote differentiation of human oligodendrocyteprecursor cells (OPC) was also evaluated. As with rat OPCs, Li81 Fab andLi81 (agly) had a dramatic effect on the human OPC cultures and resultedin the formation of highly differentiated, mature oligodendrocytes asevidenced by increases in the length of cell processes and the presenceof abundant myelin sheet structures that are stained by the anti-MBPantibody. The number of human OPCs that were MBP+ after treatment with acontrol antibody (hIgG1) or Li81 (agly) is shown in FIG. 3. Only a smallpercentage of less differentiated oligodendrocytes was seen in thecontrol antibody (hIgG1) treated cells (FIG. 3). Similar results wereobtained using Li81 Fab.

Example 6 Li81 Promotes Remyelination in Lysolecithin-Treated Brains

The cerebellar slice culture system is an in vitro model for analyzingmechanisms of remyelination. Coronal cerebellar slices from P17 ratsapproximately 300 μm thick were placed in tissue culture medium for 4days, then treated with lysolecithin for 24 hours to inducedemyelination and incubated with medium containing Li81 (agly) (30, 10,3, and 1 μg/ml) or an isotype-matched control antibody (5c8) for 3 daysto allow remyelination to occur. Remyelination was visualized by blackgold immunostaining, which selectively stains myelin in brain slices. Inblack gold stained sections, myelinated white matter appears dark brownand demyelinated lesions appear as pale brown or white.

Treatment of the brain slices with lysolecithin resulted in almostcomplete demyelination of the tissue as evidenced by loss of staining inthe control antibody treated culture. Li81 (agly) treatment resulted inrobust remyelination as evidenced from the reappearance of the staining.The immunohistochemistry data were quantified by measuring the intensityof the black gold staining as summarized in the bar graph of FIG. 4.Treatment with Li81 (agly) resulted in approximately a 30-fold increasein myelinated tissue over the level seen in the control treated brainslice. The overall level of remyelination following Li81 (agly)treatment was approximately half of that observed without anydemyelination treatment. Similar results were obtained using Li81 Fab.

Example 7 Decreased Binding of Aglycosylated Anti-LINGO-1 Antibody toFc(Gamma) Receptors

Relative binding affinities of IgG for human Fc receptors (CD16, CD32aand b, CD64) were measured using the Amplified Luminescent ProximityHomogeneous Assay (ALPHA) technology from Perkin Elmer. The assay wasperformed in a competitive format in which serial dilutions of testantibodies were incubated with the receptor-GST fusion proteins andanti-GST acceptor beads overnight at 40° C. in a 96-weft plate.Streptavidin donor beads and biotinylated wild-type IgG1 were alsoincubated overnight at 40° C. in a separate tube and then added to theassay plate the next day. The plates were incubated at room temperaturefor 2 hours with gentle shaking and read in an Envision plate reader(Perkin Elmer). The data were plotted to a 4-parameter curve fit usingGraphpad Prism software to calculate the IC₅₀ values in order todetermine the relative binding affinities. The antibodies tested wereLi81 (agly), an isotype-matched control antibody (5c8) and anaglycosylated version of the control antibody. The data are plotted inFIG. 5. The IC₅₀ values of Li81 (agly) were calculated as follows:CD32a: 365 μg/mL (down 60× from wt), CD32b: 350 μg/mL (down 15× fromwt), CD16:179 μg/mL (down 50× from wt), and CD64: >100 μg/mL (down >100×from wt).

The ability of Li81 (agly) to bind certain Fc(gamma) receptors was alsoevaluated in a cell bridging assay. For these studies, CHO cellsexpressing human LINGO-1 were plated into 96-well tissue culture plates,then incubated with serial dilutions of test samples, and with BCECF-AMlabeled U937 cells that naturally express both CD64 (FcgR1) and CD32(FcgRIIa). Bound U937 cells were quantified by fluorescence(ex485/em530) using a cytofluor plate reader. Anti-LINGO-1 monoclonalantibodies Li33 and Li13 that bind LINGO-1 with nM EC₅₀ values andcontain wild type Ig1 frameworks showed typical sigmoidal binding curveswith EC₅₀ values of 0.17 and 0.23 μg/mL, respectively (FIG. 6). Incontrast, Li81 (agly) showed a very poor bridging response, consistentwith a reduction in the affinity of the aglycosylated framework for CD64and CD32. The shift in dose response is consistent with a >10 fold dropin binding. Control huIg1 showed no bridging activity.

Example 8 Aglycosylated Anti-LINGO-1 Antibody does not PromoteComplement Activation

The effect of the aglycosylated variant of IgG1 antibodies on reducingC1q binding and activation of the complement pathway are welldocumented. To verify these effects on the Li81 (agly) antibody, theantibody was tested for C1q binding in an ELISA format and forcomplement-dependent cytotoxicity (CDC) in CHO cells expressing humanLINGO-1. For the CDC assay LINGO-1 and Lt-beta (positive control)expressing CHO cells were treated with serial dilutions of anti-LINGO-1antibodies or LtbetaR-Fc, low toxicity rabbit serum complement andpropidium iodide and assayed for killing. Li81 (agly) did not elicit acytotoxic response whereas the LtbetaR-Fc reagent promoted a robustkilling response (FIG. 7). To date no measurable cytotoxic response inthe CDC assay has been observed with any LINGO-1 targeted reagentincluding Li33 or Li13 as intact Ig1 anti-LINGO-1 Mabs (shown in FIG. 7)or aggregated 1A7 Ig1.

Example 9 Anti-LINGO-1 Antibodies Promote Myelination In Vivo inLysolecithin Assay

The lysolecithin (LPC)-induced demyelination model is a simple in vivosystem for investigating remyelination. LPC was injected into the dorsalcolumn of 9 week old adult female Sprague Dawley rats (250 g) on day 0.Demyelination occurred within a few hours following LPC treatment. Li81(agly) or a control antibody was administered IP on day 3. The animalswere sacrificed on day 9, and the region of the spinal cord encompassingthe lesion was excised and sectioned.

Sections from control antibody-treated animals showed large lesions withextensive areas of demyelination as evident from the absence of stain inthe lesion area. Smaller lesions were apparent in Li81 (agly)-treatedrats and the lesions contained lace-like structures representative ofthe remyelinated axons (FIG. 8). In subsequent studies, the model wasrun with Li81 (agly) at 2, 1, and 0.3 mg/kg. The 2 and 1 mg/kg doses ofLi81 (agly) were highly efficacious, while effects from the 0.3 mg/kgtreated animals were less efficient. These results demonstrate thatanti-LINGO-1 antibodies promote myelination in vivo in a dose dependentmanner.

In a similar experiment, lysolecithin-treated rats are administered 2,1, and 0.3 mg/kg of Li62 (agly) antibody on day 3 instead of Li81(agly). Animals are sacrificed on day 9, and the region of the spinalcord encompassing the lesion is excised and sectioned. Sections areanalyzed to compare lesion size and myelination in animals treated withLi62 (agly) to lesion size and myelination in animals treated with acontrol antibody.

Example 10 Anti-LINGO-1 Antibodies Promote Myelination In Vivo inMOG-EAE Assay

Myelin oligodendrocyte glycoprotein (MOG)-induced murine experimentalautoimmune encephalomyelitis (EAE) is a widely accepted model forstudying the clinical and pathological features of multiple sclerosisand has been described in more detail in PCT/US2008/000316, filed Jan.9, 2008, which is incorporated by reference herein. Li81 (agly) wastested in the EAE model to determine if inhibition of endogenous LINGO-1function promotes functional recovery.

Adult 9-week-old brown Norway female rats (150 g) were injected with 75μg recombinant rat MOG (amino acids 1-125) in PBS. Animals developedsigns of EAE at 15 days. Li81 (agly) treatment or isotype control (3mg/kg) was injected IP at days 15, 18, 21, 24 and 27 (10 rats pergroup). The EAE clinical score was measured daily for 2 weeks. As shownin FIG. 9, Li81 (agly) promotes functional recovery in this model byimproving hind limb and tail movement.

In a similar experiment, MOG-treated rats are injected with Li62 (agly)or an isotype control at days 15, 18, 21, 24 and 27. The EAE clinicalscore is measured daily for 2 weeks to assess hind limb paralysis,complete tail paralysis and distal tail paralysis, and the paralysis inanimals treated with Li62 (agly) is compared to paralysis in animalstreated with the control antibody.

Example 11 Testing the Effect of LINGO-1 Antibodies and FragmentsThereof on Oligodendrocytes in an In Vivo Cuprizone Model

In order to determine if Li62, Li81, and variants thereof promotemyelination in vivo adult mice are fed cuprizone (0.2% milled withground mouse chow by weight) for 6 weeks to induce demyelination withinthe corpus callosum according to the method described by Morell P etal., Mol Cell Neurosci. 12:220-7 (1998) and in PCT/US2008/000316, filedJan. 9, 2008, which is incorporated herein by reference in its entirety.Briefly, an anti-LINGO-1 Li62 or Li81 monoclonal antibody, Fab, or avariant thereof, is stereotactically injected into the demyelinatingcorpus callosum at weeks 2, 2.5, and 3 weeks of cuprizone feeding.Control mice are stereotactically injected at the same intervals withsterilized media containing control antibody. After the 6 weeks ofcuprizone feeding is completed, the mice are returned to a normal dietfor 2, 4 and 6 weeks to allow remyelination.

The animals receiving anti-LINGO-1 antibody treatment are evaluated formature oligodendrocyte survival (based on CC1 antibody staining) andaxon myelination by IHC using anti-MBP protein antibody or luxol fastblue. CC1 antibody-positive oligodendrocytes are quantitated at fourweeks and six weeks. Increased CC1 and/or MBP levels indicate that theantibodies promote mature oligodendrocyte survival and axon myelination.

Example 12 Testing the Effect of LINGO-1 Antibodies and FragmentsThereof on Retinal Ganglion Cell Survival in the Optic Nerve TransectionModel

Anti-LINGO-1 antibodies are tested in an optic nerve transection model,which investigates factors that affect neuronal function. The rightoptic nerve of an adult rat is transected intraorbitally 1.5 mm from theoptic disc. A piece of gelfoam soaked with 6% Fluoro-Gold (FG) isapplied to the newly transected site right behind the optic disc tolabel the surviving retinal ganglion cells (RGCs). The animals aredivided into three groups which receive Li81 or Li62 monoclonalantibodies, Fabs, variants thereof, a control antibody, or PBS, byintravitreal injection. The volume of each intravitreal injection is 4μl while the dosage of each injection is 2 μg. The intravitrealinjections are performed immediately after the optic nerve transection.

All animals are allowed to survive for 1 week. Two days beforesacrificing the animals, the left optic nerve of each animal istransected and 6% FG is administered as described above to label thesurviving RGCs, to serve as the internal control. Animals are sacrificedwith an overdose of Nembutal and the retinas are dissected in 4%paraformaldehyde. Four radial cuts are made to divide the retinas intofour quadrants (superior, inferior, nasal and temporal). The retinas arethen post-fixed in the same fixative for 1 hour before they areflat-mounted with the umunting medium (Dako). The slides are examinedunder a fluorescence microscope using an ultra-violet filter (excitationwavelength=330-380 nm). Labeled RGCs are counted along the median lineof each quadrants starting from the optic disc to the peripheral borderof the retina at 500 μm intervals, under an eyepiece grid of 200×200μm². The percentage of surviving RGCs resulting from each treatment isexpressed by comparing the number of surviving RGCs in the injured eyeswith their contra-lateral eyes. Effective antibodies show increasedneuronal survival when compared to control-antibody or PBS treatedanimals.

Example 13 Testing LINGO-1 Antibodies for Remyelination in the OpticNerve Crush Model

The right optic nerve is completely crushed by #5 forceps for 10 secondsaround 1.5 mm behind the eyeball intraorbitally just beforeadministration of 2 μl of Li62 or Li81 monoclonal antibody, Fab, or avariant thereof, in 2 ml by intravitreal injection.

The animals receive a second intravitreal injection of the sametreatment one week after the surgery. Two weeks after the surgery, theanimals are perfused with EM fixatives, postfixed and processed forsemithin and ultrathin sections. The longitudinal optic nerve sectionsare stained and prepared for myelin observation. The myelination of theproximal and the distal parts of the crushed optic nerve are comparedamong different treatment groups. Animals treated with Li62 or Li81monoclonal antibody, Fab, or variants thereof will be analyzed forremyelination in the distal part of the optic nerve compared to thecontrols.

Example 14 Testing LINGO-1 Antibodies for Axon Regeneration in the OpticNerve Crush Model

The right optic nerve is crushed by #5 forceps for 10 seconds around1.5-2 mm behind the eyeball intraorbitally just before administration of2 μg of Li62 or Li81 monoclonal antibody, Fab, or a variant thereof inPBS via intravitreal injection. Control animals are administered acontrol antibody or PBS. The animals receive a second intravitrealinjection of the same treatment one week after the surgery. Three daysprior to sacrifice of the test animals (day 11 of the experiment), 2 mlof CTB-FITC is injected intravitreally to label, anterograde, theregenerative optic nerve axons. On the 14th day post surgery, theanimals are perfused and postfixed. The crushed optic nerve is processedfor frozen longitudinal sections. The CTB-FITC labeled axons, whichcross the lesion site are counted as regenerative fibers at variousdistances beyond the crush site. The regeneration of axons in animalstreated with Li62 or Li81 monoclonal antibody, Fab, or a variant thereofand compared to control animals.

Example 15 Identification and Characterization of Li113

Li62 variant C02, also called Li113, was elected for further study. ALINGO-1 ELISA assay demonstrated that the Li113 Fab bound to LINGO-1with an EC50 of 0.09 nM. The EC50 measurements of the Li33 Fab, the Li62Fab, and Li81 Fab were 0.30 nM, 0.26 nM and 0.11 nM, respectively in thesame experiment (FIG. 10). The Li113 Fab was also tested in theoligodendrocyte differentiation, assay. The assay was performedessentially as described above in Example 2, but MBP levels weremeasured by ELISA. The results for a control monoclonal antibody, theLi81 monoclonal antibody, the Li62 Fab, and the Li113 Fab are shown inFIG. 11. These data demonstrate that Li113 can effectively bind toLINGO-1 and promote oligodendrocyte differentiation.

Example 16 Isotype Switching to Improve Antibody Solubility

The anti-LINGO-1 Li33 Fab was converted into a full human antibody andexpressed in mammalian cells. Three different IgG frameworks (Ig1, Ig2,and Ig4) were evaluated both in wildtype and aglycosyl forms. For theIg2 framework, the V234A/G237A mutation was also evaluated as analternative to the glycosylation site mutation to eliminate FcRIIabinding. Native human kappa light chain and heavy chain signal peptideswere used to direct secretion of Li33 light and heavy chains,respectively, in mammalian cell hosts. The variable domain fragment ofthe light chain was subcloned into a shuttle vector containing theintact signal peptide and light chain kappa chain constant region. Thevariable domain fragment of the heavy chain was subcloned into shuttlevectors containing the intact signal peptide and Ig1, Ig1agly, Ig4,Ig4agly, Ig2, Ig2agly, and Ig2 V234A/G237A heavy chain constant region.

Each of the generated antibodies showed typical antibody features bySDS-PAGE gel analysis under both reducing and non-reducing conditions.In addition, the ability of each of the isotypes to bind LINGO-1 wasassessed in an ELISA format. ELISA plates were coated with LINGO-1,treated with serial dilutions of each antibody, and bound Li33 wasdetected with an alkaline phosphatase anti-human Fab conjugate. Theseven Mabs showed similar EC50 values for binding to LINGO-1 (Table 7)with apparent affinities of 0.12 nM for the Ig1 wt and agly, ˜0.24 nMfor Ig2 and Ig2agly, and ˜0.36 nM for Ig4 and Ig4agly.

TABLE 7 Impact of Li33 Ig frameworks on solubility. Solubility LINGO-1binding Stability ° C. SEC Li33 Isotype (mg/mL) EC50 (nM) TM1 TM2 %monomer Ig1 0.9 0.12 69 76 99 Ig1agly 0.3 0.12 60 77 >99 Ig4 >30 0.35 6472 98 Ig4Pagly 0.3 0.37 56 73 95 Ig2 >50 0.23 69 76 96 Ig2agly 0.2 0.2659 76 98 Ig2-V234A/ 5.6 0.19 69 76 95 G237A Ig1 Fab2 0.3 0.10 — 77 98Ig2 Fab2 >50 0.39 — 77 98 Ig1 Fab >50 0.68 — 76 95 PEG-Fab >50 1.9 — 7798 Ig1agly >40 0.12 55 75 98 reduced Ig1 reduced >50 0.15 63 75 98 Ig1pH 7.0 0.9 0.08 68 77 Ig1 pH 6.5 1.7 0.10 69 77 Ig1 pH 6.0 2.4 0.10 6978 Ig1 pH 5.5 30 0.16 66 81 Ig1 pH 5.0 >50 0.45 66 81 Ig1 pH 4.5 >50 2.162 82 Ig1 pH 4.0 >50 16 54 78 Ig1 pH 3.5 >50 34 46/66 74 Ig1 pH 3.0 >50ND 34/52 72

The solubility of the isoforms was assayed as follows. Samples werebuffer exchanged using multiples cycles of concentration and dilution incentrifugation YM30 filter devices. Protein concentration was determinedimmediately after concentration from absorbance scans and again after 5days at 4° C. following filtration through a 0.45 μm filter. If theabsorbance had decreased, samples continued to be monitored over time at4° C. When possible, samples were concentrated to 50 mg/mL; however,some of the purified constructs were only concentrated to the amountindicated due to small sample size.

All three of the aglycosyl antibodies had poor solubility at pH 7.0 withextensive precipitation at concentrations greater than 0.3 mg/mL Mab.The solubility of the Ig1wt Mab was slightly improved (0.9 mg/mL) whilethe Ig2 and Ig4 Mabs were soluble at the highest concentration tested.The solubility of the Ig2 Mab was >50 mg/mL, representing a >150 foldincrease over the aglycosyl version of the same construct. The Ig2V234A/G237A variant was intermediate in terms of its solubility. Belowthe solubility limits, the antibodies were stable to prolonged storageat 4° C. and to freeze-thaw.

Since the solubility of a protein can be significantly reduced at pHnear its isoelectric point (pI), pI values for the Li33 Mabs weredetermined by isoelectric focusing. Samples were subjected toisoelectric focusing on a pH 3-10 IEF minigel (Invitrogen).Elecrophoresis was carried out at 100V for 1 hour, 200V for 1 hour and500V for 30 minutes. The gel was fixed, stained with Coomassie brilliantblue R-250, and destained. All of the antibodies had basic isoelectricpoints with pI values >pH 8.2. The pI values for both the Ig1 andIg1agly Li33 were ˜9.0, for Ig4 and Ig4agly Li33 were 8.2, and for Ig2,Ig2agly, and Ig2 V234A/G237A were 8.5.

The aggregation state of the antibodies was studied by size exclusionchromatography (SEC). SEC was performed on a Superdex 200 FPLC columnusing 20 mM sodium phosphate pH 7.2 and 150 mM NaCl as the mobile phase.The column was run at 0.3 mL/min. The column effluent was monitored byUV detection at 280 nm, and purity was assessed by peak height. Allconstructs eluted as a single prominent peak with an apparent molecularmass of 150 kDa with >95% purity (Table 7). Selected profiles are shownin FIG. 12. The soluble fraction for the Ig1agly by SEC was >99% monomerwith no evidence of soluble aggregates. In contrast, the Ig2 contained2% dimer and 2% higher molecular mass aggregates. The aggregation stateof Li33 Ig2 was further evaluated by analytical ultracentrifugation(FIG. 13), which revealed that the antibody actually formed reversibledimers at high concentrations. Thus, while the Ig2 framework preventedthe transition to an insoluble aggregate, it had not ablated allprotein-protein interactions.

The stability of a protein can also impact its solubility. The thermalstability of the constructs was measured by differential scanningfluoremetry (DSF). Measurements were conducted on an Mx3005p real-timePCR system (Agilent Technologies) in a 96-well format using 10 μg ofprotein in 50-55 μL phosphate buffer (at neutral pH) supplemented withSYPRO orange fluorophor (Invitrogen) at a final concentration of 10×.Samples were heated from 25° C. to 95° C. at 1° C./min with fluorescenceintensity measured 3 times every 1° C. Fluorescence intensities wereplotted as a function of temperature. Melting temperatures (Tm) werederived from these curves by taking the negative derivative (“−R′(T)” inthe Mx3005p software) and selecting the local minima of the derivativeplots. For DSF measurements at various pH values, 10 mM sodium citratewas used as the buffering agent.

Tin values for the Fab region (1M2) were 76-77° C. for the Ig1 and Ig2constructs and 72-73° C. for the Ig4 wt and agly. TM1 values for the CH2region were variable. Transitions were 8-10° C. lower for each of theagly constructs. The Ig4 constructs were the least stable. The stabilityof Li33 Ig1 and Li33 Ig2 was also studied using guanidine denaturationas an alternative to thermal denaturation to assess stability. FIG. 14.The denaturation curve for the Ig1 was monophasic with a transitionpoint of 3.1 M guanidine. The denaturation curve for the Ig1 Fab wassimilar to that for the intact Mab. Reduction of the Ig1 Fab shifted thetransition point to 1.8 M guanidine. Li33 Ig2 denatures at a higherguanidine concentration than the Ig1 with a 50% transition point of 4.1M. The shape of the curve suggests there may be several transitions.

To further assess features of the antibodies that were affectingsolubility a variety of conditions and fragmentation were tested (Table7). Fab2 fragments of Ig1 and Ig2 were generated enzymatically withpepsin, and a Fab fragment of Ig1 was generated with papain. Thesolubility of the Ig2 Fab was >50 mg/mL, whereas the solubility of theIg1 Fab2 was only 0.3 mg/mL. The solubility of the Ig1 Fab was >50mg/mL.

A pegylated version of the Fab was also generated. For pegylation, Ig1Fab2 at 1.2 mg/mL in 40 mM sodium borate pH 7.0 and 0.1 mM TCEP wasincubated for 75 min at 37° C. The reduced sample was desalted on a G25Mcolumn that had been equilibrated in 5 mM MES pH 5.0 and 50 mM NaCl(final Fab concentration 0.5 mg/mL). After storage overnight at 4° C.,over 90% of the disulfide bond holding the heavy and light chaintogether had reoxidized to a Fab′ leaving the 2 hinge Cys residues freefor conjugation. 10 kDa methoxy-polyethyleneglycol maleimide (PEGmal)(Nektar) was added to 0.4 mg/mL, and MES pH 6.0 was added to 25 mM. Thesample was incubated at room temperature for 2.5 hours and overnight at4° C. The sample was then subjected to cation exchange chromatography ona Fractogel EMD sulfate column. The column was washed with 2 columnvolumes of 10 mM sodium phosphate pH 6.0, and the PEG-Fab was elutedwith 10 mM sodium phosphate pH 6.0 and 50 mM NaCl. The pegylated versionalso had excellent solubility (Table 7).

Fragmentation had little impact on stability. No TM1 signal was observedfor the Fab2 and Fab moieties as expected, since the TM1 transition isproduced from the CH2 domain. Differences in LINGO-1 binding wereconsistent with the nature of the products as the three monvalentversions had reduced binding. Reduction of the interchain disulfidesthat link the heavy-heavy and heavy-light chains also had a verydramatic affect on solubility. After reduction, the Li33 Ig1 and Ig1agly Mabs were soluble at the highest concentration tested (Table 7).Reduction had only a slight effect on thermal stability and had noimpact on aggregation state or LINGO-1 binding.

Finally, the effect of pH on solubility was tested. A dramatictransition in the solubility of Li33 Ig1 occurred between pH 6 and pH5.5. Below pH 5.5 the protein was very soluble, and above pH 5.5, it hadpoor solubility. Thermal stability and LINGO-1 binding were reducedunder the more acidic conditions that had improved solubility.

Example 17 Disulfide Bond Mapping

The disulfide structure of Li33 Ig2 was determined by peptide mapping.In these experiments, alkylation of Li33 Ig2 was done under denaturingand non-reducing conditions. 5 uL of 100 mM idoacetamide solution wasadded to 25 μL of the solution containing ˜22.5 μg of the protein, and25 mg of guanidine hydrochloride was immediately added to the solution.The solution was kept at room temperature in the dark for 30 minutes.The alkylated proteins were recovered by precipitation in cooledethanol. The solution was stored at −20° C. for 1 hour and thencentrifuged at 20,000 g for 12 min at 4° C. The alkylated and recoveredproteins were digested with 20% (w/w) of endo-Lys-C in 2 M urea and 0.6M Tris-HCl pH 6.5 for 8 hours at room temperature. Then 5% (w/w) oftrypsin was added to the solution, and the solution was kept overnightat room temperature. Another aliquot of 5% of trypsin was added thesecond morning, and the solution was kept at room temperature for anadditional 4 hours. Prior to analysis of the digests, 50 μL of freshlyprepared 8 M urea was added to the digest, and the solution was splitinto two parts: one was analyzed after reduction, which was done byincubating the digest with 40 mM DTT at 37° C. for 1 hour; the otherpart was not reduced before analysis. The reduced and non-reduceddigests were analyzed on an LC-MS system comprised of a reversed-phaseHPLC (Alliance, Waters Milford Mass.) and an LCT mass spectrometer(Waters Corp., Milford, Mass.). The separation was carried out on a 1.0mm×15-cm Vydac C4 column (214TP5115) using a flow rate of 0.07 mL/min.The mobile phase A was water with 0.03% trifluoroacetic acid, and mobilephase B was acetonitrile with 0.024% trifluoroacetic acid. The gradientwas running linearly from 0 to 15% B in 65 minutes, then to 26% B in 55minutes, then to 39% B in 30 minutes. The ESI source voltage was set at3,300 V, and the cone voltage was 30 V, with a desolvation temperatureset to 200° C. Peaks on the maps were identified using MassLynx 4.1software.

Unlike Ig1 and Ig4 Mabs, which each contain a distinct interchaindisulfide pattern, the disulfide structure on an Ig2 antibody is complexand contains a mixture of different isoforms. The detected disulfidelinked peptide clusters for Li33 Ig2 are listed in Table 8.

TABLE 8 Disulfide structure of Li33 IgG2. Calc. Mass Detected Mass RTRecovery Linkage (Da) (Da) (min) (%)* Non-hinge IntrachainLT2(C1)1-LT6(C2) 4204.89 4204.90 133.1 100% LT11(C3)-LT18(C4) 3555.753555.73 123.1 100% HT2(C1)-HT10(C2) 3442.55 3442.54 141.5 100%HT14(C4)-HT15(C5) 8073.92* 8073.78* 142.3  90% HT21(C10)-HT26(C11)3986.85 3986.84 124.5 100% HT34(C12)-HT39(C13) 3844.82 3844.77 110.7100% Interchain LT20(C5)-HT13(C3) 1535.69 1535.66 83.5  30% HingePattern HT19(C6-9)-HT19(C6-9) 5350.56 5350.52 138.9 10 A + AHT19(C6-9)-HT19′(C6-9) 5125.40 5125.37 140.5 HT19′ (C6-9)-HT19′(C6-9)4900.26 4900.25 142.3 Pattern 2x(HT13(C3) + LT20(C5)) + 8425.62*8425.46* 138.2 20 B + B 2xHT19(C6-9) 2x(HT13(C3) + LT20(C5)) + 8200.33*8200.02* 139.4 HT19(C6-9) + HT19′(C6-9) 2x(HT13(C3) + LT20(C5)) +7975.04* 7975.05* 140.8 2xHT19′(C6-9) Pattern HT13(C3) + LT20(C5) +6885.24 6885.47 138.8 15 A + B 2xHT19(C6-9) HT13(C3) + LT20(C5) +6660.08 6660.29 140.1 HT19(C6-9) + HT19′(C6-9) HT13(C3) + LT20(C5) +2xHT19′(C6-9) 6434.92 6434.95 141.5 Pattern C HT13(C3) + LT20(C5) +4209.97 4209.88 138.2  5 HT19(C6-9) HT13(C3) + LT20(C5) + 3984.823984.78 141.7 HT19′(C6-9) *indicates average mass

All the predicted intrachain disulfides were detected with highrecovery. The recovery percentage of the intrachain disulfide linkagebetween the 3rd cysteine on the heavy chain to the 5th cysteine on thelight chain was 30%. Four different forms of disulfide linkage weredetected for the hinge region. The first form is the classic fourparallel linked disulfides in the hinge region. The second form is the3rd cysteines in both heavy chains and the 5th cysteines in both lightchains linked to cysteines in the diner of the hinge peptide. The thirdform is a mixture of the first and second forms, on one arm, the 3rdcysteine in the heavy chain linked to the 5th cysteine in the lightchain, whereas, one the other arm, the 3rd cysteine in the heavy chainand the 5th cysteine in the light chain are linked to cysteines in thedimer of the hinge peptide. The forth form is a half antibody with the3rd Cys in the heavy chain and the 5th cysteine in the light chainforming disulfide bonds with cysteines in the hinge. Furtherheterogeneity in the Li33 Ig2 Mab resulted from glycosylation. Typicalfor a glycosylated protein, 15 different glycan structures wereobserved. Glycans were largely simple bianternary core structures (G055%, G1 24%, G2 3%). Six percent of the glycan structures weresialyated.

Example 18 Targeted Mutagenesis to Improve Solubility

The crystal structures of Li33 Ig1 Fab and Li33 Ig2 Fab2 were determinedin order to identify contact points that could be altered to improvesolubility.

The crystal structure of the Li33 Ig1 Fab was solved to 3.2 Å. Li33 Ig1Fab at 5 mg/ml was mixed at a volumetric ratio of 1:1 with a reservoirsolution consisting of 2 M ammonium sulfate, 0.1M sodium acetate pH 3.5,and 0.1 M TCEP. Football-shaped crystals were grown by vapor diffusionat 20° C. They were then cryoprotected by transferring them into 2Mammonium sulfate, 0.1 M citrate pH 3.5, 20% glycerol, 10% sucrose, and10% xylitol for 2 minutes and freezing them by a quick transfer intoliquid nitrogen. The crystals diffracted to 3.2| at the SGXcat beamlineat the Advanced Photon Source (Argonne, Ill.). Data processing with theHKL program package v. 1.97 [1] revealed the crystals to belong to aP6(5)22 space group with approximate cell dimensions a, b=90.6 Å,c=215.0 Å, and a=b=90 o, g=120o. The crystal structure was solved bymolecular replacement based on another IgG1 homology model (AQC2 mutantFab PDBID: 2B2X) in PHASER (Otwinowski and Minor, Methods in Enzymology276: 307-326 (1997)) with all possible arrangements of the screw accessleading to a clear solution in space group P6(5)22. Model building ofthe single Fab and 4 sulfates in Coot 0.5.2 (Vagin et al., ActaCrystallogr D Biol Crystallogr 60:2184-2195 (2004)) followed byrefinement using Refmac5 (Emsley and Cowtan, Acta Crystallogr D BiolCrystallogr 60:2126-32 (2004)) to 3.2 Å resolution resulted in a finalR-factor of 19.3% and Rfree of 28.9% with reasonable geometry (Table1).

For crystallization of the Li33 Ig2 Fab2, the sample at 7.2 mg/ml wasmixed at a volumetric ratio of 1:1 with a reservoir solution consistingof 12% Peg3350, 0.1M phosphate citrate pH 4, and 0.2M NaCl. Rod-shapedcrystals were grown by vapor diffusion at 20° C. They were thencryoprotected by transferring them into 20% Peg3350, 0.1M phosphatecitrate pH 4, 0.2M NaCl, and 15% glycerol for 2 minutes and thenfreezing them by a quick transfer into liquid nitrogen. The crystals ofthe Li33 Ig2 Fab2 diffracted to 2.8 Å at the SGXcat beamline at theAdvanced Photon Source (Argonne, Ill.). Data processing with the HKLprogram package v. 1.97 (Otwinowski and Minor, Methods in Enzymology276: 307-326 (1997)) revealed the crystals to belong to a P1 space groupwith approximate cell dimensions α=91.7, β=109.5A, c=118.4, and α=61.4o,β=74.3o, γ=87.6o. The crystal structure was solved by molecularreplacement based on another Ig2 homology model (3GIZ) in PHASE k.

These crystal structure provided a unique opportunity to identifycontact points and use rational design to address solubility issues.FIG. 15 shows structural interfaces from the crystal structure withCDR-CDR and CDR-framework contact points highlighted. Five residues wereidentified with intermolecular contacts, W50, W94, W104, I57, and P54and are highlighted in the figure. Targeted site directed mutagenesiswas performed on the key residues within the CDR sequences thatcontributed to contact points. Results from selected mutations are shownin Table 9.

TABLE 9 Impact of targeted mutagenesis on Li33 solubility. LINGO-1 SECSolubility binding Stability ° C. % Li33 Isotype (mg/mL) EC50 (nM) TM1TM2 monomer Ig1agly 0.3 0.12 60 77 >99 Ig1aglyW94V/I57V >10 0.15 5974/82 99 Ig1aglyW94V/I57S >7 0.38 58 75/82 99 Ig1aglyW94V/I57P >8 0.0758 75/83 99 Ig1aglyW94V/I57T >7 0.13 59 75/83 99 Ig2 >50 0.23 69 76 96Ig2agly 0.3 0.26 59 76 98 Ig2-V234A/G237A 5.6 0.19 69 76 95+94V/104Q/57S >50 2.9 67 74 95 +94V/104Q/57A >50 2.4 69 73 95

The series of Ig1 agly W94VI57 mutations all improved solubility with noimpact on LINGO-1 binding, stability, and level of aggregation detectedby size exclusion chromatography. The Ig2-PDL W104QW94VI57 mutationsalso improved solubility with no impact on stability and aggregation,but the additional mutation caused a 10-fold loss in LINGO-1 bindingaffinity. The triple mutants were further characterized by analyticalultracentrifugation where there was no evidence for dimer formation.

Example 19 PeEGylated Li33 Fab

PEGylated Li33 Fab constructs were created both by enzymatic digestionof the Li33 Mab and by direct expression of the Fab.

In order to directly express a Li33 Fab, a Fab construct was geneticallyengineered from the Li33 Ig1 construct so that the heavy chainterminated at P231 in the hinge, thereby deleting the Fc moiety andproviding a single, unpaired cysteine from the natural Ig1 hingesequence that could be targeted for PEGylation. The light chain sequencewas not altered. The amino acid sequence for the heavy chain of Li33Fab′, as predicted from the DNA sequence, is:

(SEQ ID NO: 146)EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWVRQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAREGHNDWYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP.The amino acid sequence for the light chain as predicted from the DNAsequence is:

(SEQ ID NO: 145)DIQMTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYDKWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

The Li33 Fab construct was expressed in CHO cells. Cells expressing highlevels of the Li33 Fab were selected by FACS sorting. The Li33 Fabcontains 11 cysteines: 5 that form disulfides and a single freecysteine. Of these, only the disulfide that holds the heavy and lightchains together and the free cysteine are surface exposed and potentialtargets for PEGylation. To verify the reactivity of these cysteines, theLi33 Fab was reduced with 0.1 mM TCEP, treated with an excess ofPEGmaleimide (PEGmal) (0.2 mM), and analyzed by SDS-PAGE for rapidassessment of both the extent of reduction and PEGylation.

Reduction (step 1 in methods 1, 2, and 3 of FIG. 16) was performed asfollows. To 1 mL of Protein A purified Fab′ at 1.2 mg/mL, 40 μl, of 1Msodium borate, pH 8.4, and 1 μL of 100 mM TCEP (final 0.1 mM) wereadded, and the sample was incubated for 75 minutes at 37° C. Under theseconditions, greater than 90% of the product was routinely reduced toheavy and light chain when analyzed by non-reducing SDS-PAGE. Thepredominant products after reduction were free heavy and light chainthat each migrated with an apparent mass of 25 kDa.

For PEGylation with 20 kDa methoxy-polyethyleneglycol maleimide (PEGmal)(Nektar) (step 2 in method 1 of FIG. 16), MES pH 6.0 was added to 25 mMfrom a 0.5 M stock solution and PEGmal was added to 0.4 mg/mL (2× molarexcess) from a 20 mg/mL stock solution that was stored at −70° C. Thesample was incubated at room temperature for 2.5 hours then overnight at4° C. and then subjected to cation exchange chromatography on aFractogel EMD sulfate (EM Merck) resin.

When the TCEP-reduced Fab was treated with PEGmal, a nearly completeloss of the free heavy and light chains was observed. This observationis consistent with modification of the three cysteines. Three new bandswere detected under non-reducing conditions that correspond to heavychain or light chain containing a single PEG, heavy chain containing twoPEGs, and the PEGylated Fab with a single PEG attached (FIG. 17, method1).

The experiment was repeated with 5 kDA, 10 kDA, and 40 kDA PEGmal, andthe same three bands were present regardless of the size of the PEGmalthat was utilized. However, their molecular weights varied in a mannerthat was consistent with the size of the attached PEG moiety. Forexample the mono-PEGylated heavy or light chain had apparent molecularweights of 35, 40, 50, and 100 kDa when the samples were treated withthe 5, 10, 20, and 40 kDa PEGs, respectively, and the di-PEGylated heavychain migrated at 45, 80, 100, and 250 kDa under the same conditions.The PEGylated Fab bands with a single 5, 10, 20, and 40 kDa PEGattached, migrated with masses of 70, 80, 90, and 150 kDa, respectively.

Under reducing conditions the prominent bands corresponding to heavychain or light chain containing a single PEG or two PEGs were notaffected, while the PEG-Fab bands disappeared. A new prominent bandcorresponding to free heavy or light chain was present after reduction.In all cases, the percent of product that was accounted for in thePEG-Fab band was only 20% of the starting material indicating that thepredominant product was Fab with three PEGs attached. These resultsconfirm that after reduction, all three of the reactive cysteines areaccessible for modification. Subsequent studies have focused on 20 kDaPEG as the reactive group.

Follow-up studies was performed to optimize the PEGylation reaction inorder to form the desired PEG-Fab product (methods 2 and 3 in FIG. 16).The results are shown in FIG. 17 (methods 2 and 3). In both studies theTCEP reductant was removed on a desalting column prior to reaction ofthe reduced Fab′ with the PEG. In method 2, the PEG was addedimmediately after the removal of the TCEP, allowing both the PEGylationreaction and oxidation of the interchain disulfide to occurconcurrently. In contrast, for method 3, oxidation of the interchaindisulfide was allowed to occur prior to the addition of the PEG. Bothmethods resulted in a much higher percentage of the desired PEG-Fabproduct than when PEGylation was performed in the presence of TCEP(method 1). When oxidation and PEGylation occurred simultaneously(method 2), the major contaminants were PEGylated light chain anddi-PEGylated heavy chain. When oxidation and PEGylation were performedsequentially (method 3), the major contaminants were unmodified Fab andFab2. The PEG-Fab′ was purified from the reaction mixture by cationexchange chromatography on a Fractogel EMD sulfate resin. Theunpegylated reaction products from the later scheme can be more easilyfractionated away from the PEG-Fab based on preliminary purificationstudies.

In order to create a PEGylated Li33 Fab construct by enzymaticdigestion, Fab2 fragments of Li33 were first generated with pepsin.Samples were dialyzed overnight at 4° C. against 10 mM sodium acetate pH3.6. Pepsin was added at an enzyme:protein ratio of 1:100 and incubatedat 37° C. for 6 hours for complete conversion of the Mab to Fab2. The pHof the digest was adjusted to 7.5 with 200 mM Hepes and the sample wasloaded onto a Protein A Sepharose column at 10 mg protein/mL resin. Thecolumn was washed with 5 column volumes of PBS, 4 column volumes of 25mM sodium phosphate pH 5.5 and 100 mM NaCl, and the Fab2 was eluted fromthe resin with 10 mM sodium citrate pH 3.3 and 50 mM NaCl, collectingsix 0.5 column volume steps. The pH of the samples was adjusted to 4.7with NaOH. Peak fractions were pooled, filtered through 0.22μ units,aliquoted and stored at −70° C.

For PEGylation, Ig1 Fab2 at 1.2 mg/ml in 40 mM sodium borate pH 7.0 and0.1 mM TCEP was incubated for 75 minutes at 37° C. The reduced samplewas desalted on a G25M column that had been equilibrated in 5 mM MES pH5.0 and 50 mM NaCl (final Fab concentration 0.5 mg/ml). After storageovernight at 4° C., over 90% of the disulfide bonds holding the heavyand light chains together had reoxidzed to a Fab′ leaving the 2 hingeCys residue free for conjugation. 10 kDa PEGma1 (Nektar) was added to0.4 mg/mL and MES ph 6.0 was added to 25 mM. The sample was incubated atroom temperature for 2.5 hours, overnight at 4° C., and then subjectedto cation exchange chromatography on a Fractogel EMD sulfate column. Thecolumn was washed with 2 column volumes of 10 mM sodium phosphate pH6.0, and the PEG-Fab was eluted with 10 mM sodium phosphate pH 6.0 and50 mM NaCl. The results are shown in FIG. 18.

When evaluated for function by ELISA, the PEG-Li33 Fab′ product wasfully active in its ability to bind LINGO-1.

Example 20 Pegylated Li81 and Li113

Li81 fragments were created by pepsin cleavage and subject to bothC-terminal and N-terminal PEGylation.

For N-terminal PEGylation, a Fab was generated from the Li81 antibody byenzymatic digestion with papain and repurification. Li81 Fab at ˜2 mg/mLin 10 mM sodium citrate pH 6.0, 5 mg/mL 20 kDamethoxy-polyethyleneglycol proprionaldehyde (Nektar), and 5 mM sodiumcyanoborohydride were incubated at room temperature for 24 hours. The pHwas adjusted to pH 4, and the samples were concentrated to 10 mg Fab/mLand subjected to cation exchange chromatography at room temperature on aFractogel EMD sulfate column (Merck) at 10 mg Fab/mL resin. The columnswere washed with 2.5 column volumes of 10 mM sodium citrate pH 4.7, and1 column volume of 10 mM sodium citrate pH 4.7, 15 mM NaCl. The PEG-Fabwas eluted with 10 mM sodium citrate pH 4.7, 50 mM NaCl, and 0.25 columnvolume fractions were collected. Fractions were analyzed by SDS-PAGE andpeak fractions containing monopegylated Fab were pooled, filtered,aliquoted and stored at −70° C. Protein concentrations were estimatedfrom absorbance at 280 nm using the theoretical extinction coefficientsfor the Fabs.

For C-terminal PEGylation, Fab2 fragments of Li81 Ig1agly were generatedwith pepsin. Samples were dialyzed overnight at 4° C. against 10 mMsodium acetate pH 3.6. Pepsin was added at an enzyme:protein ratio of1:1000 and incubated at 37° C. for 3 hours to achieve completeconversion of the Mab to Fab2. The pH of the digest was adjusted to 7.5with 200 mM Hepes, and the sample was loaded onto a Protein A Sepharosecolumn at 10 mg protein/mL resin. The column was washed with 5 columnvolumes of PBS, 4 column volumes of 25 mM sodium phosphate pH 5.5 and100 mM NaCl, and the Fab2 was eluted from the resin with 10 mM sodiumcitrate pH 3.3, 50 mM NaCl, collecting 6×0.5 column volume steps. The pHof the samples were adjusted to 4.7 with NaOH. Peak fractions werepooled, filtered through 0.22μ units, aliquoted, and stored at −70° C.

For pegylation, Li81 Ig1agly Fab2 at 2.6 mg/mL in 20 mM sodium borate pH7.0, 0.2 mM TCEP was incubated for 90 minutes at 37° C. The reducedsample was diluted with 2 volumes of 10 mM sodium citrate pH 4.7 andloaded onto a Fractogel EMD sulfate column (10 mg Fab′/mL resin),pre-equilibrated in the citrate pH 4.7 buffer. The column was washedwith 3 column volumes of 10 mM sodium citrate pH 4.7, 2.5 column volumesof 10 mM sodium phosphate pH 6.0, 50 mM NaCl, and the Fab′ was elutedwith 7×0.8 column volume steps of 10 mM sodium phosphate pH 6.0, 200 mMNaCl. The protein eluate was diluted with water to a final proteinconcentration of 1.4 mg/mL. After storage for 48 hours at 4° C., most ofthe disulfide bond holding the heavy and light chain together hadreoxidized to a Fab′ leaving the 2 hinge Cys residues free forconjugation. 10 kDa methoxy-polyethyleneglycol maleimide (PEGmal)(Nektar) was added to 1.0 mg/mL, and sodium citrate pH 6.5 was added to10 mM. The sample was incubated at room temperature for 2.5 hours andthen overnight at 4° C., concentrated and buffer exchanged in an AmiconUltra-15 30K centrifugal filter device to 8 mg/mL with a final bufferconcentration of 10 mM citrate pH 4.7, 6 mM NaCl, and then subjected tocation exchange chromatography on a Fractogel EMD sulfate column (8 mgprotein/mL resin), pre-equilibrated in the citrate pH 4.7 buffer. Thecolumn was washed with 1 column volume of 10 mM sodium citrate pH 4.7buffer, and the PEG-Fab′ was eluted with 12 0.15 column volume steps of10 mM sodium citrate pH 4.7, 50 mM NaCl, 2 0.15 column volume steps of10 mM sodium citrate pH 4.7, 100 mM NaCl, and 7 0.15 column volume stepsof 10 mM sodium citrate pH 4.7, 200 mM NaCl. Column fractions wereanalyzed by SDS-PAGE. Peak fractions were pooled and the buffer adjustedto 15 mM sodium citrate pH 6.5, 125 mM NaCl. Protein concentration wasdetermined by absorbance at 280 nm. The sample was filtered through a0.22μ unit, aliquoted and stored at −70° C.

The N-terminally PEGylated Li81 Fab2 demonstrated an EC50 of 0.15 ng/ml,while the C-terminally PEGylated Li81 Fab′ demonstrated an EC50 of 0.054ng/ml, but both showed equivalent activity in an oligodendrocyteproliferaction assay (0.1 μg/ml).

Li113 was also N-terminally PEGylated by following the same protocol asdescribed for Li81 N-terminal PEGylation. The FACS results shown in FIG.19 demonstrate that PEGylated Li81 and Li113 can bind to LINGO-1.

Example 21 Evaluation of Functional Properties of LINGO-1 AntibodyVariants

The efficacy of Li81 antibodies and fragments thereof was evaluated inan ELISA assay to asses LINGO-1 binding (FIG. 20), in an oligodendrocyteproliferation assay (“OPC assay”) (FIG. 21), and in a rat remyelinationassay (FIG. 22). The results demonstrate that each of the Li81 Mab,Fab2, Fab, N-PEG-Fab, and C-PEG Fab binds to LINGO-1 and is functionalin in vitro assays. The biochemical and in vitro properties of themolecules tested are summarized in Table 10.

TABLE 10 Biochemical and in vitro LINGO-1 antibody properties. PropertyMab Fab2 Fab N-PEG C-PEG Valency Bivalent Bivalent Monovalent MonovalentMonovalent Fc Low None None None None fucntion effector; Wt FcRn Size140 100 50 250 250 SEC (kDa) EC50 0.017 0.017 0.041 0.15 0.054 (nM) OPC0.1 0.1 0.1 0.1 0.1 assay (μg/ml)

Example 22 LINGO-1 antibody and antibody fragment thermal stability

Thermal denaturation was carried out using a UV-visiblespectrophotometer fitted with a computer-controlled,thermoelectrically-heated cuvette holder. Solutions were equilibrated at25° C. for 15 minutes in a 200:1 microcuvette. The temperature of thecuvette holder was then ramped from 25° C. to 90° C. at a rate of 2°C./min, and the denaturation of the protein was followed by continuousmonitoring of absorbance at 280 nm. The mid-point of the cooperativeunfolding event, Tm, was obtained from the melting curves by determiningthe temperature at which the measured absorbance was mid-way between thevalues defined by lines extrapolated from the linear regions on eachside of the cooperative unfolding transition. The results ofdenaturation experiments are shown in FIG. 23 and demonstrate that theTm of the LINGO-1 antibodies and Fabs tested range from about 66° C. toabout 76° C.

Example 23 Li33 Variants

In order to identify other Li33 variants with improved affinity, thevariants listed in Tables 11 below, were constructed and tested. Adirect binding ELISA assay was performed using LINGO-Fc coated plates.The half maximal concentration that gave 50% saturation of binding wasmeasured and is reported as a ratio of the same measurement for Li33. Inaddition, the OD450 for maximal saturation value was measured and isreported as a ratio of the same measurement for Li33.

TABLE 11 Li33 Variants. Chain with ELISA Plateau Variant Variant SEQ IDNO Ratio Ratio WT Li33 NA (WT) 145 and 146 1.0 0.91 W50H Heavy 147 1.30.87 W50F Heavy 148 1.2 0.86 W50L Heavy 149 1.8 1.00 W50M Heavy 150 2.00.93 P53L Heavy 151 2.2 1.13 P53S Heavy 152 2.5 1.43 P53T Heavy 153 2.10.85 P53W Heavy 154 3.9 0.55 W104V Heavy 155 2.8 0.94 W104H Heavy 1560.1 0.91 W104S Heavy 157 2.7 0.85 W104Q Heavy 158 2.4 0.91 I57G Heavy159 2.3 0.91 I57M Heavy 160 3.8 1.09 I57N Heavy 161 2.9 1.10 I57H Heavy162 3.2 1.07 I57L Heavy 163 0.7 1.00 I57F Heavy 164 2.0 0.83 W94A Light165 1.6 0.91 W94D Light 166 1.5 0.87 W94L Light 167 0.6 0.97 W94N Light168 0.8 1.05 W94G Light 169 0.9 1.03 W94Q Light 170 0.9 1.05 W94V Light171 1.4 0.91 W94S Light 172 1.3 0.99 W50G Heavy 173 2041.4 21.79 W50IHeavy 174 93.5 12.44 W50D Heavy 175 4.0 0.71 W104M Heavy 176 3.8 0.88W104L Heavy 177 2.5 0.69 W104T Heavy 178 3.5 0.33 W104I Heavy 179 6.50.62 P53G Heavy 180 3.1 0.83 I57W Heavy 181 0.0 I57Y Heavy 182 0.0 I57SHeavy 183 0.6 0.96 I57P Heavy 184 0.6 1.27 I57V Heavy 185 1.4 1.13 I57THeavy 186 1.1 1.01 W104Q Heavy 187 1.1 0.67

Several variants with an affinity within 2 fold of Li33 and a plateauvalue of at least 85% of that of Li33 were identified: W50F, W50L, W50M,I57L, I57F, W94A, W94L, W94N, W94G, W94Q, W94V, W94S, I57S, I57P, I57V,and I57T.

Li62 Variants Example 24

In order to identify other Li62 variants with improved affinity, thevariants listed in Table 12 below, are constructed and tested. A directbinding ELISA assay is performed using LINGO-Fc coated plates Variantsthat show an affinity within 2 fold of Li62 and a plateau value of atleast 85% of that of Li62 are identified and analyzed in in vitro and inviro functional assays as described above.

TABLE 12 Li62 Variants Chain with Variant Variant SEQ ID NO Li62 NA (WT)1 and 9 W50H Heavy 188 W50F Heavy 189 W50L Heavy 190 W50M Heavy 191 P53LHeavy 192 P53S Heavy 193 P53T Heavy 194 P53W Heavy 195 W104V Heavy 196W104H Heavy 197 W104S Heavy 198 W104Q Heavy 199 I57G Heavy 200 I57MHeavy 201 I57N Heavy 202 I57H Heavy 203 I57L Heavy 204 I57F Heavy 205W50G Heavy 206 W50I Heavy 207 W50D Heavy 208 W104M Heavy 209 W104L Heavy210 W104T Heavy 211 W104I Heavy 212 P53G Heavy 213 I57W Heavy 214 I57YHeavy 215 I57S Heavy 216 I57P Heavy 217 I57V Heavy 218 I57T Heavy 219W104Q Heavy 220

Li81 Variants Example 25

In order to identify other Li81 variants with improved affinity, thevariants listed in Table 13 below, are constructed and tested. A directbinding ELISA assay is performed using LINGO-Fc coated plates. Variantsthat show an affinity within 2 fold of Li81 and a plateau value of atleast 85% of that of Li81 are identified and analyzed in in vitro and inviro functional assays as described above.

TABLE 13 Li81 Variants Chain with Variant Variant SEQ ID NO Li81 NA (WT)5 and 13 M96L Light 221 M96I Light 222 M96Q Light 223 M96K Light 224M96A Light 225 M96V Light 226 M96Y Light 227 M96F Light 228 P53L Heavy229 P53S Heavy 230 P53T Heavy 231 P53W Heavy 232 P53G Heavy 233 W94ALight 234 W94D Light 235 W94L Light 236 W94N Light 237 W94G Light 238W94Q Light 239 W94V Light 240 W94S Light 241

Li113 Variants Example 26

In order to identify other Li113 variants with improved affinity, thevariants listed in Table 14 below, are constructed and tested. A directbinding ELISA assay is performed using LINGO-Fc coated plates. Variantsthat show an affinity within 2 fold of Li113 and a plateau value of atleast 85% of that of Li113 are identified and analyzed in in vitro andin vino functional assays as described above.

TABLE 14 Li113 Variants Chain with Variant Variant SEQ ID NO Li113 NA(WT) 66 and 9 W50H Heavy 242 W50F Heavy 243 W50L Heavy 244 W50M Heavy245 P53L Heavy 246 P53S Heavy 247 P53T Heavy 248 P53W Heavy 249 W104VHeavy 250 W104H Heavy 251 W104S Heavy 252 W104Q Heavy 253 I57G Heavy 254I57M Heavy 255 I57N Heavy 256 I57H Heavy 257 I57L Heavy 258 I57F Heavy259 W50G Heavy 260 W50I Heavy 261 W50D Heavy 262 W104M Heavy 263 W104LHeavy 264 W104T Heavy 265 W104I Heavy 266 P53G Heavy 267 I57W Heavy 268I57Y Heavy 269 I57S Heavy 270 I57P Heavy 271 I57V Heavy 272 I57T Heavy273 W104Q Heavy 274

The present invention is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the invention, and any compositions or methodswhich are functionally equivalent are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and accompanying drawings.Such modifications are intended to fall within the scope of the appendedclaims.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1-62. (canceled)
 63. An isolated polynucleotide comprising a nucleicacid encoding an immunoglobulin light chain variable region (VL),wherein the CDR1, CDR2, and CDR3 regions of said VL comprise thesequences of SEQ ID NOs: 10, 11 and 12, and wherein an antibody orantigen-binding fragment thereof comprising said VL specifically bindsto LINGO-1. 64-222. (canceled)
 222. The polynucleotide of claim 63,wherein the VL comprises the sequence of SEQ ID NO:9.
 223. A vectorcomprising the polynucleotide of claim
 63. 224. A vector comprising thepolynucleotide of claim
 222. 225. The polynucleotide of claim 63,wherein the nucleic acid encoding the VL is a cDNA.
 226. Thepolynucleotide of claim 222, wherein the nucleic acid encoding the VL isa cDNA.
 227. A host cell comprising the polynucleotide of claim 63,wherein the host cell further comprises a nucleic acid encoding animmunoglobulin heavy chain variable region (VH), wherein the CDR1, CDR2,and CDR3 regions of said VH comprise the sequences of SEQ ID NOs: 2, 3and
 4. 228. A host cell comprising the polynucleotide of claim
 222. 229.A host cell comprising the vector of claim 223, wherein the host cellfurther comprises a nucleic acid encoding a VH, wherein the CDR1, CDR2,and CDR3 regions of said VH comprise the sequences of SEQ ID NOs: 2, 3and
 4. 230. The host cell of claim 229, wherein the nucleic acidencoding the VH is present in the same vector as the nucleic acidencoding the VL.
 231. The host cell of claim 229, wherein the nucleicacid encoding the VH is present in separate vector than the nucleic acidencoding the VL.
 232. A host cell comprising the vector of claim 224.233. The host cell of claim 232, wherein the host cell further comprisesa nucleic acid encoding a VIA comprising the sequence of SEQ ID NO:1.234. The host cell of claim 233, wherein the nucleic acid encoding theVH is present in the same vector as the nucleic acid encoding the VL.235. The host cell of claim 233, wherein the nucleic acid encoding theVH is present in separate vector than the nucleic acid encoding the VL.236. A method of producing an anti-LINGO-1 antibody or anantigen-binding fragment thereof, comprising culturing the host cell ofclaim 227, and recovering said antibody or antigen-binding fragment.237. A method of producing an anti-LINGO-1 antibody or anantigen-binding fragment thereof, comprising culturing the host cell ofclaim 229, and recovering said antibody or antigen-binding fragment.238. A method of producing an anti-LINGO-1 antibody or anantigen-binding fragment thereof, comprising culturing the host cell ofclaim 233, and recovering said antibody or antigen-binding fragment.239. The polynucleotide of claim 63, wherein the antibody orantigen-binding fragment that specifically binds to LINGO-1 is arecombinant antibody.
 240. The polynucleotide of claim 222, wherein theantibody or antigen-binding fragment that specifically binds to LINGO-1is a recombinant antibody.